Secretion Of Proteoglycans Research Articles

A neutrophil-derived proteolytic inactive elastase homologue (hHBP) mediates reversible contraction of fibroblasts and endothelial cell monolayers and stimulates monocyte survival and thrombospondin secretion.

Human heparin-binding protein (hHBP) is a recently discovered proteolytically inactive neutrophil elastase homologue with sequence identity to azurocidin and CAP37. The protein has antibacterial properties and chemotactic activity toward monocytes. In the present work, we show that monocytes, cultured under serum-free conditions, developed morphological changes and formed multicellular aggregates 4 h after the addition of hHBP at a concentration of 10 micrograms/ml. However, after prolonged incubation (11 days) with unchanged medium, the cells spread again. The hHBP-treated cells had a two- to threefold increase in survival compared to control cells, measured using trypan blue as an indicator of living cells. Differentiation of the alive cells to macrophages was detected by changes in morphology, a threefold increase in protein content, and a three- to fourfold increase in acid phosphatase activity. When monocytes in parallel experiments were labelled with [35S]methionine de novo synthesis and secretion of thrombospondin in a dose-dependent manner was observed after 16 h, with half-maximal secretion at 2 micrograms hHBP/ml and a maximal 12-fold increase in secretion with respect to controls at 16 micrograms/ml. Supplementary labeling with [35S]sulfate revealed that the same monocytes down-regulated the secretion of a large proteoglycan (300-400 kd), apparently also with a half-maximal decrease rate at 2 micrograms/ml hHBP. Exposure of confluent fibroblast and endothelial cell monolayers to hHBP (10 micrograms/ml) in the absence of fetal calf serum resulted in cell contraction leaving gaps between cells, the phenomenon being recognizable within 4 h after addition of hHBP. Addition of fetal calf serum to a concentration of 10% completely restored the monolayers. A unique role of hHBP in host defense involving recruitment of monocytes and a key function of hHBP in neutrophil extravasation in response to inflammatory chemotactic signals such as leukotriene B4, complement peptide C5a, and N-formyl-methionyl-leucyl-phenylalanine are suggested.

Inhibition of synthesis of rat parotid secretory proteoglycan in a gland slice system

The chondroitin sulphate contained within the secretory granules of the rat parotid gland and its saliva was shown to be in the form of a proteoglycan by using inhibitors of proteoglycan synthesis in a gland slice system. Gland slices were incubated in either p- nitrophenyl-β- d- xyloside or chlorate in the presence of both [ 3H]-leucine and [ 35S]-sulphate. The slices were next homogenized and either the 250 g supernatant fraction (for initial experiments) or secretory granule-containing fractions were isolated. Protein and proteoglycans of these fractions were precipitated in 10% trichloracetic acid (TCA), and glycosaminoglycans in cetylpyridinium chloride. [ 3H]-leucine and [ 35S]-sulphate were quantitated in each type of precipitate by scintillation counting. The results showed that 1 mM xyloside had no effect on protein or glycosaminoglycan synthesis but blocked incorporation of radiosulphate into TCA-precipitable material. Sixteen mM chlorate almost totally inhibited incorporation of radiosulphate into glycosaminoglycan and TCA-precipitable material. These findings demonstrate that the rat parotid secretory chondroitin sulphate is indeed a proteoglycan because its synthesis is blocked by the protein-core analogue acceptor, p- nitrophenyl-β- d- xyloside . This system offers opportunities for exploring the functional role of chondroitin sulphate proteoglycan in this salivary gland.

The dependency of collagen fibrillogenesis in vitro on fibroblast culture conditions. Fibroblasts in mono- and multi-layers.

The extracellular matrix produced by monolayer and tridimensional cultures of fibroblasts was investigated using histochemical and ultrastructural methods. In monolayer cultures, collagen and proteoglycans produced by fibroblasts could not be organized into morphologically recognizable structures. Tridimensional fibroblast cultures produced a well organized matrix with periodic, parallel ordered collagen fibrils of 50 nm diameter, criss-crossed by alcianophylic segments 6-10 nm thick in diameter and 100-300 nm in length, parallel to each other, perpendicular to the collagen fibrils and spaced 67 nm from each other. Some alcianophylic segments lay perpendicular to the above described ones, with maximum lengths of 65-70 nm. Alcianophylic segments are the ultrastructural evidence of structural proteoglycans. These observations suggest that the culture conditions influence the collagen and proteoglycans secretion, so that the final organization of the matrix results quite different.

Polarized rat uterine epithelium in vitro: responses to estrogen in defined medium.

Previously described procedures for the culture of immature rat uterine epithelium (UE) allowed the cells to proliferate to confluence and develop morphological and functional polarity. The present study describes the transition from culture of UE cells in serum to a serum-free defined medium. This was accomplished with no significant alteration in the ability of UE cells to attain morphological and functional polarity. In defined medium, which contained estrogen (2.5 x 10(-9) M), UE cells proliferated to confluence, demonstrated separation of apical and basal plasma membrane domains, and displayed preferential secretion of proteins and proteoglycans from the apical surface. Apical secretions of polarized cultures contained complement component C3, the secreted portion of the immunoglobulin A receptor and the secretory glycoprotein, USP-1. The cell surface adhesion molecule, CAM 105, could be demonstrated at the apical cell surface. Expression of this profile of secretory and cell surface markers, representative of the in vivo estrogen response of immature rat UE cells, correlated with an in vitro state of non-receptivity of polarized UE cells toward blastocysts which remained viable and competent to attach. We conclude that the polarized UE cell that develops in the described defined medium expresses a phenotype similar to that which characterizes the in vivo uterine estrogen response.

Polarized rat uterine epithelium in vitro: responses to estrogen in defined medium

Previously described procedures for the culture of immature rat uterine epithelium (UE) allowed the cells to proliferate to confluence and develop morphological and functional polarity. The present study describes the transition from culture of UE cells in serum to a serum-free defined medium. This was accomplished with no significant alteration in the ability of UE cells to attain morphological and functional polarity. In defined medium, which contained estrogen (2.5 x 10(-9) M), UE cells proliferated to confluence, demonstrated separation of apical and basal plasma membrane domains, and displayed preferential secretion of proteins and proteoglycans from the apical surface. Apical secretions of polarized cultures contained complement component C3, the secreted portion of the immunoglobulin A receptor and the secretory glycoprotein, USP-1. The cell surface adhesion molecule, CAM 105, could be demonstrated at the apical cell surface. Expression of this profile of secretory and cell surface markers, representative of the in vivo estrogen response of immature rat UE cells, correlated with an in vitro state of non-receptivity of polarized UE cells toward blastocysts which remained viable and competent to attach. We conclude that the polarized UE cell that develops in the described defined medium expresses a phenotype similar to that which characterizes the in vivo uterine estrogen response.

A heterotrimeric G protein, G alpha i-3, on Golgi membranes regulates the secretion of a heparan sulfate proteoglycan in LLC-PK1 epithelial cells.

A heterotrimeric G alpha i subunit, alpha i-3, is localized on Golgi membranes in LLC-PK1 and NRK epithelial cells where it colocalizes with mannosidase II by immunofluorescence. The alpha i-3 was found to be localized on the cytoplasmic face of Golgi cisternae and it was distributed across the whole Golgi stack. The alpha i-3 subunit is found on isolated rat liver Golgi membranes by Western blotting and G alpha i-3 on the Golgi apparatus is ADP ribosylated by pertussis toxin. LLC-PK1 cells were stably transfected with G alpha i-3 on an MT-1, inducible promoter in order to overexpress alpha i-3 on Golgi membranes. The intracellular processing and constitutive secretion of the basement membrane heparan sulfate proteoglycan (HSPG) was measured in LLC-PK1 cells. Overexpression of alpha i-3 on Golgi membranes in transfected cells retarded the secretion of HSPG and accumulated precursors in the medial-trans-Golgi. This effect was reversed by treatment of cells with pertussis toxin which results in ADP-ribosylation and functional uncoupling of G alpha i-3 on Golgi membranes. These results provide evidence for a novel role for the pertussis toxin sensitive G alpha i-3 protein in Golgi trafficking of a constitutively secreted protein in epithelial cells.

Disruption of microtubules alters polarity of basement membrane proteoglycan secretion in epithelial cells

Pages C691–C700: J. B. De Almeida and J. L. Stow. “Disruption of microtubules alters polarity of basement membrane proteoglycan secretion in epithelial cells.” Because of the disappointing reproduction of the halftones, this article is reprinted at the end of the July issue.

Disruption of microtubules alters polarity of basement membrane proteoglycan secretion in epithelial cells.

Basement membrane proteins such as the heparan sulfate proteoglycan (HSPG) are secreted in a polarized fashion from the basolateral membrane of epithelial cells. We have used the microtubule-disrupting drug colchicine to study the role of the microtubule network in directing constitutive secretion to the basolateral membrane of LLC-PK1 renal epithelial cells. Microtubule depolymerization induced by colchicine resulted in fragmentation and redistribution of fluorescently labeled trans-Golgi membranes. Increased immunofluorescent staining of HSPG was associated with these dispersed Golgi cisternae. The biosynthetic processing of HSPG was not significantly altered by the loss of microtubules or by the dispersal of the Golgi elements. The most striking effect of microtubule disruption was the loss of polarity of HSPG secretion. Immunoprecipitation studies showed that HSPG was secreted from both apical and basolateral surfaces of LLC-PK1 cells treated with colchicine, and a similar result was found for the delivery of laminin, another basement membrane protein. In contrast, there was no change in the distribution of an integral basolateral membrane protein, Na(+)-K(+)-ATPase, following colchicine treatment. Our results provide the first demonstration that microtubules are involved in the constitutive trafficking of basolateral secretory proteins. These data also suggest that there may be an inherent difference in the targeting or delivery of membrane and secretory proteins to the basolateral cell surface.

Comparison of the effects of non-steroidal anti-inflammatory drugs (NSAIDs) on proteoglycan synthesis by articular cartilage explant and chondrocyte monolayer cultures

Experiments were conducted to study proteoglycan biosynthesis by rabbit articular chondrocytes cultured in the presence of NSAIDs and 35SO 2− 4 for up to 8 days. Both articular cartilage explants and confluent chondrocyte monolayer culture models were used. Medium was changed every 2 days and the [ 35SO 4]proteoglycans which had accumulated in the medium and the extracellular matrix during the culture intervals were assayed separately. In long-term experiments, drugs were removed on day 8, and proteoglycan production during a 10–12 day culture interval also was assayed. The drugs studied were diclofenac, indomethacin, ketoprofen, piroxicam and tiaprofenic acid, at concentrations of 0, 0.1, 1, 10, 50 and 100μg/mL. Whereas proteoglycan production by cell cultures was maximal early in the culture period, explants produced more proteoglycans as time progressed. The highest concentrations of all of the drugs, especially diclofenac and indomethacin, inhibited proteoglycan secretion by both cell and explant cultures. However, after removal of the drugs from the cultures, suppressed proteoglycan production reversed to levels equivalent to, or higher than controls in the cell cultures, but largely persisted in explant cultures. About 70–80% of proteoglycans produced by explants were retained in the matrix, whereas about 80–90% of proteoglycans produced by cell cultures were secreted into the medium. Where drugs inhibited proteoglycan production, the levels were reduced by approximately the same proportions in both extracellular matrix and culture medium fractions. Of the NSAIDs examined only ketoprofen demonstrated a stimulatory effect on PG synthesis in explant cultures at a physiological concentration (0.1μg/mL).

Disruption of microtubules alters polarity of basement membrane proteoglycan secretion in epithelial cells

Basement membrane proteins such as the heparan sulfate proteoglycan (HSPG) are secreted in a polarized fashion from the basolateral membrane of epithelial cells. We have used the microtubule-disrupting drug colchicine to study the role of the microtubule network in directing constitutive secretion to the basolateral membrane of LLC-PK1 renal epithelial cells. Microtubule depolymerization induced by colchicine resulted in fragmentation and redistribution of fluorescently labeled trans-Golgi membranes. Increased immunofluorescent staining of HSPG was associated with these dispersed Golgi cisternae. The biosynthetic processing of HSPG was not significantly altered by the loss of microtubules or by the dispersal of the Golgi elements. The most striking effect of microtubule disruption was the loss of polarity of HSPG secretion. Immunoprecipitation studies showed that HSPG was secreted from both apical and basolateral surfaces of LLC-PK1 cells treated with colchicine, and a similar result was found for the delivery of laminin, another basement membrane protein. In contrast, there was no change in the distribution of an integral basolateral membrane protein, Na+-K+-ATPase, following colchicine treatment. Our results provide the first demonstration that microtubules are involved in the constitutive trafficking of basolateral secretory proteins. These data also suggest that there may be an inherent difference in the targeting or delivery of membrane and secretory proteins to the basolateral cell surface. polarized secretion; heparan sulfate proteoglycans; sorting; Golgi processing Submitted on July 10, 1990 Accepted on November 12, 1990

Independent secretion of proteoglycans and collagens in chick chondrocyte cultures during acute ascorbic acid treatment

The mechanisms regulating the secretion of proteoglycans and collagens in chondrocytes, in particular those operating at the level of the rough endoplasmic reticulum (RER), are largely unknown. To examine these mechanisms, I studied the effects of acute ascorbate treatment on the secretion of two collagen types (types II and IX) and two proteoglycan types (PG-H and PG-Lb, the major keratan sulphate/chondroitin sulphate proteoglycan and the minor chondroitin sulphate proteoglycan respectively in cartilage) in scorbutic cultures of chick vertebral chondrocytes. I found that the scorbutic chondrocytes synthesized underhydroxylated precursors of types II and IX collagen that were secreted very slowly and accumulated in the RER. When the cultures were treated acutely with ascorbate, both macromolecules underwent hydroxylation within 1-1.5 h of treatment, and began to be secreted at normal high rates starting at about 2 h. Proteoglycan synthesis and secretion, however, remained largely unaffected by ascorbate treatment. Both the half-time of newly synthesized PG-H core protein in the RER and its conversion into completed proteoglycan were unchanged during treatment. Similarly, the overall rates of synthesis and secretion of both PG-H and PG-Lb remained at control levels during treatment. The data indicate that secretion of types II and IX collagen is regulated independently of secretion of PG-H and PG-Lb. This may be mediated by the ability of the RER of the chondrocyte to discriminate between procollagens and proteoglycan core proteins.

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.

Neutrophil elastase and cathepsin G stimulate secretion from cultured bovine airway gland serous cells.

To investigate the hypothesis that neutrophil proteases stimulate airway gland secretion, we studied the effect of human cathepsin G and elastase on secretion of 35S-labeled macromolecules from cultured bovine airway gland serous cells. Both proteases stimulated secretion in a concentration-dependent fashion with a threshold of greater than or equal to 10(-10) M. Elastase was more potent than cathepsin G, causing a maximal secretory response of 1,810 +/- 60% over baseline at 10(-8) M. The maximal response to cathepsin G (1,810 +/- 70% over baseline at 10(-7) M) was similar to the maximal response to elastase. These responses were greater than 10-fold larger than the response to other agonists such as histamine. Protease-induced secretion was noncytotoxic and required catalytically active enzymes. The predominant sulfated macromolecule released by proteases was chondroitin sulfate proteoglycan. Immunocytochemical staining demonstrated chondroitin sulfate in cytoplasmic granules and decreased granular staining after stimulation of cells with elastase. The neutrophil proteases also degraded the proteoglycan released from serous cells. Cathepsin G and elastase in supernatant obtained by degranulation of human peripheral neutrophils also caused a secretory response. Thus, neutrophil proteases stimulate airway gland serous cell secretion of chondroitin sulfate proteoglycan and degrade the secreted product. These findings suggest a potential role for neutrophil proteases in the pathogenesis of increased and abnormal submucosal gland secretions in diseases associated with inflammation and neutrophil infiltration of the airways.

Turnover of proteoglycans by chick lens epithelial cells in cell culture and intact lens.

Chick lens epithelial cells were cultured on plastic and type IV collagen substrata, and the confluent cultures were labeled continuously with [35S]sulfate for 20 h. Intact lenses were also labeled in the same way. 35S-Proteoglycans isolated from those cultures were compared for their molecular sizes and glycosaminoglycan compositions. The results have shown that: 1) Proteoglycans synthesized by cells on type IV collagen were significantly smaller than those by cells on plastic. 2) Proteoglycans of intact lens showed a broad distribution of molecular size and contained a high proportion of chondroitin sulfate in the medium fraction compared to those of the two cell cultures. In order to explain such differences between proteoglycans from cultures, label-chase experiments with [35S]sulfate were done for proteoglycans synthesized. 35S-Proteoglycans isolated at each chase time 0, 2.5, and 17 h) were compared and the following results were found: 1) The cell layers of both "plastic" and "type IV collagen" cultures contained glycosaminoglycan species predominantly at each chase time rather than proteoglycans. 2) Changes in the glycosaminoglycan compositions of medium fractions of cell cultures were observed during the chase period; in medium of the "plastic" culture, proteoheparan sulfate increased with chase time, whereas in medium of the "type IV collagen" culture, chondroitin sulfate glycosaminoglycan (not proteoglycan) increased with chase time. 3) In intact lens culture, lens capsule fraction at every chase time contained a proteoglycan unique in molecular size, which was not found in cell culture fractions. 4) All fractions from intact lens cultures contained a higher content of chondroitin sulfate at every chase time than the respective fractions from cell cultures. These results suggest that adhesion of the cells to type IV collagen or lens capsule influences the degradation and secretion of proteoglycans. In addition, they can account partially for the above-described differences in molecular sizes and glycosaminoglycan compositions between 35S-proteoglycans from various cultures continuously labeled with [35S]sulfate.

Heparin induces changes in the synthesis of porcine aortic endothelial cell heparan sulfate proteoglycans

Heparin is known to bind to cultured endothelial cells. This report documents that addition of heparin to endothelial cells results in an alteration of the heparan sulfate proteoglycan synthetic pattern. Specifically, the addition of saturating amounts of heparin to confluent cultures of porcine aortic endothelial cells results in an increase in the amount of radiolabeled heparan sulfate proteoglycan secreted into the growth medium. The increase is apparent as early as 8 h after heparin administration. Although there is often a decrease in the amount of cell surface heparan sulfate proteoglycan produced, it is not sufficient to account for the increase in the secreted form. Of the other glycosaminoglycans tested, only dextran sulfate and commercial heparan sulfate induce changes in heparan sulfate proteoglycan synthesis and secretion. Chondroitin sulfate glycosaminoglycans do not elicit this synthetic change. These data indicate that endothelial cells can alter the synthesis of heparan sulfate proteoglycans in response to extracellular signals including heparin and related glycosaminoglycans.

Evaluation of the effect of antiarthritic drugs on the secretion of proteoglycans by lapine chondrocytes using a novel assay procedure.

A new method is described for separating free 35SO4-- from 35SO4 labelled proteoglycans synthesised by rabbit articular chondrocytes cultured in the presence of excess 35SO4--. The procedure uses the low solubility product of barium sulphate to remove, by precipitation, free 35SO4-- from culture medium. Optimum recovery of 35SO4 labelled proteoglycans was achieved after papain digestion to release 35SO4-glycosaminoglycans, and addition of chondroitin sulphate before the precipitation step. Using this assay, we studied the effect of six drugs-indomethacin, diclofenac, sodium pentosan polysulphate, glycosaminoglycan polysulphate ester, tiaprofenic acid, and ketoprofen-on the secretion into the medium of labelled proteoglycans by lapine chondrocytes. The six drugs were tested at 0.1, 1, 10, 50, and 100 micrograms/ml over four consecutive 48 hour culture periods. A consistent concentration-response pattern was found for the four non-steroidal anti-inflammatory drugs (NSAIDs) studied. Generally they inhibited proteoglycan secretion at 50 and 100 micrograms/ml but had no effect at lower concentrations. Inhibition of secretion was strongest with indomethacin and diclofenac at 50 and 100 micrograms/ml. In contrast with the NSAIDs studied, the two sulphated polysaccharides (sodium pentosan polysulphate and glycosaminoglycan polysulphate ester) at low concentrations increased proteoglycan secretion by chondrocytes, with maximal stimulation occurring at 1 microgram/ml. Sodium pentosan polysulphate, but not glycosaminoglycan polysulphate ester, showed inhibitory activity at 50 and 100 micrograms/ml.

Vectorial secretion of proteoglycans by polarized rat uterine epithelial cells.

We have studied proteoglycan secretion using a recently developed system for the preparing of polarized primary cultures of rat uterine epithelial cells. To mimic their native environment better and provide a system for discriminating apical from basolateral compartments, we cultured cells on semipermeable supports impregnated with biomatrix. Keratan sulfate proteoglycans (KSPG) as well as heparan sulfate-containing molecules (HS[PG]) were the major sulfated products synthesized and secreted by these cells. The ability of epithelial cells to secrete KSPG greatly increased in parallel with the development of cell polarity. Furthermore, KSPG secretion occurred preferentially to the apical medium in highly polarized cultures. In contrast, HS(PG) secretion did not increase along with development of polarity, although most HS(PG) (85%) were secreted apically as well. Pulse-chase studies indicated that highly polarized cultures secreted 80-90% of the sulfated macromolecules they synthesized, predominantly to the apical secretory compartment. The half-lives for KSPG and HS(PG) secretion were approximately 3 and 4 h, respectively. Parallel studies of cells cultured on tissue culture plastic-coated with biomatrix indicated that neither the state of confluency nor the biomatrix was primarily responsible for inducing the KSPG secretion observed in polarizing cultures. Experiments with uterine strips indicated that the steroid hormone, 17-beta-estradiol, markedly stimulated synthesis and secretion of sulfated macromolecules, but had no preferential effect on KSPG production. The ratio of KSPG to HS(PG) secretion from uterine strips was similar to that found in the apical medium of highly polarized cell cultures. Thus, the pattern of proteoglycan secretion observed in polarized cell cultures mimicked that observed for uterine cells, although the preferential increase in KSPG production by polarized cells could not be attributed to an estrogen response. Collectively, these studies describe the major sulfated molecules secreted by rat uterine epithelial cells under varying conditions and provide evidence for a novel influence of cell polarity on the cell's ability to secrete sulfated glycoconjugates.

Biosynthesis of chondroitin sulfate proteoglycan by P388D1 macrophage-like cell line.

Macrophages are in large part responsible for the subendothelial deposition of lipid within the artery wall during the early stages of atherogenesis. Proteoglycans secreted by these cells may play a role in this pathological process either by trapping lipoproteins in the extracellular matrix or by enhancing the formation of lipid-laden foam cells. The synthesis and secretion of proteoglycan was studied in the P388D1 macrophage-like cell line cultured in the presence of 35S-sulfate. The radiolabeled proteoglycan had a Kd of 0.69 on Sepharose CL-2B corresponding to an Mr of 2.8 x 10(5). It consisted of approximately 13 chondroitin sulfate chains of Mr 20,000 attached to a core protein with an Mr of 18,000. The chondroitin sulfate chains contained both N-acetylgalactosamine 6-sulfate and N-acetylgalactosamine 4-sulfate residues. No disulfated N-acetylgalactosamine residues were present. The P388D1 proteoglycan bound specifically to immobilized human low density lipoprotein. These results suggest that, in the focal regions of the arterial wall in which macrophages are found during the development of fatty streaks, proteoglycans secreted by these cells may affect the transport and cellular metabolism of plasma-derived lipids.

Influence of chlorate on proteoglycan biosynthesis by cultured human fibroblasts.

The influence of chlorate, an inhibitor of sulfate adenylyltransferase, on biosynthesis and secretion of proteoglycans was investigated in cultured human skin fibroblasts. At up to 10 mM concentrations, chlorate caused a reduction of [35S]sulfate incorporation into small chondroitin sulfate/dermatan sulfate proteoglycan by up to 96%. Incorporation of [3H]leucine and [3H] glucosamine was only slightly affected. No influence was seen on the polymerization degree of the polysaccharide chain as judged by gel filtration, and on the kinetics of secretion of the proteoglycan. Concomitant with reduced sulfation, however, was an increased sensitivity toward chondroitin AC lyase which suggests a diminished epimerization of D-glucuronic acid to L-iduronic acid residues. Agarose gel electrophoresis revealed that all polysaccharide chains of control chondroitin sulfate/dermatan sulfate proteoglycan exhibited a similar sulfation degree. Chlorate treatment led to the formation of polysaccharide chains of widely varying degree of sulfation, but fully sulfated chains were synthesized even in the presence of 3 mM chlorate, and sulfate-free chondroitin was not detected. Studying the effects of chlorate treatment on the synthesis of other proteoglycan types it was found that, in cell-associated galactosaminoglycans, 6-sulfation of N-acetylgalactosamine residues was less affected than was 4-sulfation. In case of heparan sulfate the synthesis of sulfamate groups was less impaired than sulfate ester formation. Nitrous acid degradation at pH 4.1 indicated the presence of unsubstituted amino groups. Chlorate treatment may be considered as a means for the production of proteoglycans with defined structural alterations.

Inhibition of cartilage proteoglycan synthesis by interleukin 1

We have investigated the mechanism of inhibition of cartilage proteoglycan by interleukin 1. Proteoglycan synthesis was inhibited using lower doses of interleukin 1 than those required to cause cartilage resorption. There was no significant effect on DNA or total protein synthesis. Gel electrophoresis showed a direct inhibitory effect on core protein synthesis while pulse-chase experiments using radiolabelled sulphate showed no alteration in the rate of intracellular transport and secretion of completed proteoglycan. Chondrocytes incubated with cycloheximide showed a first-order decrease in rate of uptake of radiolabelled sulphate ( t 1 2 = 25 mins ) but interleukin 1 induced inhibition showed a delay of at least 1 hr, consistent with a requirement to deplete intracellular pools of protein before effects on post-translational events could be observed. Foetal and neonatal cartilage responded to the cytokine in a similar way to adult cartilage.