Effects of /i-xylosides on proteoglycan biosynthesis and morphology of PC12 pheochromocytoma cells and primary cultures of rat cerebellum R. K. MARGOLIS*, B. GOOSSEN, H. TEKOTTE, L. HILGENBERG and R. U. MARGOLIS Department of Pharmacology, State University of New York, Health Science Center, Brooklyn, New York 11203, and Department of Pharmacology, New York University Medical Center, New York, New York 10016, USA • Author for correspondence at: Department of Pharmacology, Box 29, State University of New York, Health Science Center, 450 Clarkson Avenue, Brooklyn, NY 11203, USA Summary We have examined the effects of /J-xylosides, which ively). /J-Xyloside inhibition of proteoglycan biosyn- act as exogenous acceptors for glycosaminoglycan thesis was accompanied by significant morphologi- chain initiation, on the morphology and proteogly- cal effects in NGF-treated PC12 cells, consisting of an can biosynthesis of PC 12 pheochromocytoma cells, increase in length and decrease in the branching, and on monolayer, aggregate and explant cultures of diameter and adhesion to the collagen substratum of early postnatal rat cerebellum. PC 12 cells cultured the PC 12 cell processes. p-Nitrophenyl- and for 13 days in the presence of nerve growth factor 4-methylumbeUiferyl-/S-D-xylosides produced similar (NGF) and /J-xyloside, and labeled during days 11-13 effects, which were not seen with p-nitrophenyl-/J-D- with sodium [3SS]sulfate, showed an 8- to 11-fold galactoside. /J-Xylosides also produced distinct alter- increase in [3BS]sulfate-labeled macromolecules re- ations in the adhesion and morphology of monolayer, leased into the culture medium. Most of the increase aggregate, and explant cultures of early postnatal rat was accounted for by chondroitin sulfate, which was cerebellum, which occurred together with inhibition in the form of free glycosaminoglycan chains, which of chondroitin sulfate proteoglycan biosynthesis and were not acid-precipitable. The presence of /J-xylo- a decrease in glycosaminoglycan chain size. These side also led to a 65-115% increase in [36S]sulfate studies indicate that chondroitin sulfate (and prob- incorporation into cell-associated glycosaminogly- ably also heparan sulfate) proteoglycans play a cans and glycoproteins of untreated and NGF- significant role in modulating cell-cell and cell- treated PC 12 cells, respectively. /J-Xyloside treatment matrix interactions in nervous tissue development reduced the size of the chondroitin sulfate chains in and differentiation. both the cells and medium from approximately 34 000 to 10 000 Mr, but had much less effect on heparan sulfate, which decreased in size from 16000 to Key words: proteoglycans, chondroitin sulfate, /S-xylosides, 13 000-14 500 Mr (in the medium and cells, respect- PC12 cells, cell interactions. Introduction cells with fibronectin (Carbonetto et al. 1983) and the migration of amphibian neural crest cells on fibronectin There has recently been increasing interest in the roles of substrata (Perris and Johansson, 1987), respectively. It proteoglycans in various aspects of the development and has also recently been shown that sulfated glycosamino- function of nervous tissue (for a review, see Margolis and glycans modify growth factor-induced neurite outgrowth Margolis, 1989). In developing brain, there is a progressive in cultured PC12 pheochromocytoma cells (Damon et al. change in the localization of chondroitin sulfate proteogly- 1988). In one of the few studies of this type that did not cans and hyaluronic acid from extracellular to predomi- employ glycosaminoglycans, a chondroitin sulfate proteo- 3 nantly intracellular (cytoplasmic, intra-axonal and nu- glycan with a 280xl0 Afr core protein, which binds to clear) sites in specific types of neuronal and glial cells tenascin (cytotactin), has been implicated in cell—cell (Aquino et al. 1984a,6; Ripellino et al. 1988, 1989). Retina, interactions in developing chicken brain (Hoffman et al. peripheral nerve, and neural tumor cell lines are also 1988). known to synthesize chondroitin sulfate and heparan Since significant levels of free sulfated glycosaminogly- sulfate proteoglycans, which appear to be involved in can chains do not occur in most tissues, we have evaluated processes such as cell interactions, the anchorage and the effects of inhibiting proteoglycan biosynthesis in an localization of cell-surface acetylcholinesterase, and the attempt to obtain additional information concerning the binding of growth factors (Margolis and Margolis, 1989). role of proteoglycans in cell interactions during nervous Most of these studies have concerned basement membrane tissue development. Exogenous ^-xylosides have been heparan sulfate proteoglycans, although it has been widely employed for examining the contribution of reported that chondroitin sulfate, or a chondroitin sulfate proteoglycans to biological processes. Chondroitin sulfate proteoglycan, inhibits the adhesive interaction of nerve and other sulfated glycosaminoglycans are O-glycosidi- Journal of Cell Science 99, 237-246 (1991) Printed in Great Britain © The Company of Biologists Limited 1991 237 cally linked to serine residues in a core protein through a Immunobiologicals, Costa Mesa, CA; 1:300 dilution), rabbit anti- characteristic-glucuronosyl-galactosyl-galactosyl-xylosyl- mouse IgG (Sternberger-Meyer Immunocytochemicals, Jarretts- O-serine linkage region. /J-Xylosides competitively inhibit ville, MD; 1:50), and clonal mouse peroxidase-antiperoxidase the synthesis of glycosaminoglycan chains on xylosylated (Sternberger-Meyer; 1:100). Color development was carried out using diaminobenzidine/hydrogen peroxide. Cultures were coun- core protein, resulting in carbohydrate-deficient proteo- terstained using Mayer's hematoxylin solution (Sigma, St Louis, glycans that are usually accompanied by large amounts of MO) as described by Hockberger et al. (1987). free glycosaminoglycans in which chain initiation occurs on the exogenous /S-xyloside acceptor (Galligani etal. 1975; Analysis of glycosaminoglycans Schwartz, 1979). In the present study we have biochemi- Cells were washed, extracted with chloroform-methanol, and the cally demonstrated a significant inhibition by /J-xylosides lipid-free protein residue was digested with Pronase (after of chondroitin sulfate (and, to a much lesser extent, addition of unlabeled brain 'carrier') as described previously heparan sulfate) proteoglycan biosynthesis in nerve (Margolis et al. 1983). Cell protein was determined by the method growth factor-treated PC 12 pheochromocytoma cells and of Lowry et al. (1951) after dissolving a sample of cell suspension primary cultures of early postnatal rat cerebellum, and in 0.5 M NaOH. To determine the proportion of macromolecular that these biochemical effects are accompanied by distinct radioactivity in the media of xyloside-treated cells that was present as free glycosaminoglycan chains, samples of dialyzed morphological alterations. media were made 10% in trichloroacetic acid to precipitate proteoglycans. Pronase digests of cells and media were desalted by gel Materials and methods filtration on Sephadex G-15, and glycosaminoglycans were precipitated with cetylpyridinium chloride (Margolis et al. 1975). Cell culture The proportion of labeling in chondroitin sulfate and heparan Rat PC12 pheochromocytoma cells were cultured on collagen in sulfate was determined after digestion with chondroitinase ABC Eagle's Basal Diploid Medium with 0.2 nu or no added sodium (ICN ImmunoBiologicals, Costa Mesa, CA), followed by ultrafil- sulfate. Collagen preparation and coating and cell culture tration on a Centricon-10 membrane (Amicon). The molecular conditions were as described by Greene et al. (1987). Cells sizes of heparan sulfate and chondroitin sulfate were determined 1 cultured for 13 days with 2.5 S nerve growth factor (60 ngml" ) by gel filtration on Sepharose CL-6B, using the Kav values were grown in medium containing 1% heat-inactivated (30min, determined by Wasteson (1971). In the case of chondroitin sulfate, 56°C) horse serum, whereas in the absence of NGF, the medium the elution profile was calculated by subtracting heparan sulfate contained 3% horse serum and 1.5% fetal calf serum. /J-Xyloside radioactivity from that of the combined heparan sulfate+chon- treatment was for a total of 13 days in the presence of 1 mM droitin sulfate present in the total glycosaminoglycan fraction 4-methylumbelliferyl-/3-D-xyloside or p-nitrophenyl-/S-D-xyloside. (determined by two separate Sepharose CL-6B elutions under Cultures were labeled for 48 h with sodium [3*S]sulfate identical conditions). (lOO/fCiml"1), and the medium was dialyzed against 50 mM sodium sulfate followed by distilled water. PC 12 cell process Results diameters were measured on X670 photographic enlargements using an optical comparator-magnifier (Edmunds Scientific Co., Barrington, NJ) fitted with a 0.05 mm calibrated reticle. Effects of /3-xylosides on proteoglycan biosynthesis and morphology of PC 12 cells Monolayer and reaggregate cultures of early postnatal rat cerebellum were prepared in 35 mm dishes according to the /J-Xyloside treatment produced an almost 8-fold increase general procedure described by Trenkner and Sidman (1977). in [ S]sulfate-labeled macromolecules released into the Briefly, the meninges were removed from 2- to 3-day postnatal medium by NGF-treated PC12 cells cultured in 0.2 mM cerebella, which were chopped several times with fine scissors and sodium sulfate (11-fold in cells labeled without added immersed