Effect of fully sulfated glycosaminoglycans on pulmonary artery smooth muscle cell proliferation.

Fully sulfated heparin and other glycosaminoglycans, namely heparan, chondroitin, and dermatan sulfates, and hyaluronan have been prepared by using sulfur trioxide under mild chemical conditions. All these derivatives were assayed for antiproliferative activity on cultured bovine pulmonary artery smooth muscle cells (BPASMCs). No appreciable difference was found between heparin and fully sulfated heparin. Chondroitin and dermatan sulfates actually stimulated BPASMCs growth but full sulfonation made them strongly antiproliferative. Native hyaluronan was not antiproliferative but became strongly so after sulfonation. Neither acharan sulfate nor N-sulfoacharan sulfate had any antiproliferative activity. This suggests that O-sulfonation of the polysaccharide is critical for antiproliferative activity, whereas N-sulfonation of glucosamine residues is not.     

Binding and internalization of exogenous glycosaminoglycans in weakly and highly metastatic rhabdomyosarcoma cells

The fate of exogenous glycosaminoglycans in cultures of strongly (RMS 0) and weakly (RMS 8) metastatic rat rhabdomyosarcoma cells was studied. The time course and concentration dependence of binding and internalization of the radiolabeled sulfated glycosaminoglycans were determined. Weakly metastatic cells took up heparin, heparan and dermatan sulfates into their pericellular compartment at a higher rate than the strongly metastatic RMS 0 cells. The RMS 8 cells exhibited about two times more binding sites for these iduronic acid containing glycosaminoglycans, and internalized higher amounts of them than the RMS 0 cells. The uptake of the chondroitin sulfate into the peri- and intracellular compartments of both cell types was about 5-15% of that of the other glycosaminoglycans studied. The specificity of displacement of the pericellular heparin and dermatan sulfate by the unlabeled glycosaminoglycans indicates the involvement of specific structural features of the polysaccharide chains in the interactions of glycosaminoglycans with the surface of rhabdomyosarcoma cells, beside ionic forces due to the polyanionic character of the glycosaminoglycans. Heparin and heparan sulfate degradation products, mainly large oligosaccharides, were recovered from the surface of RMS 0 cells but were absent on the surface of the RMS 8 cells. About 30% of the internalized heparin and heparan sulfate was present in the partially degraded form in both cell types. Oligosaccharides derived from glycosaminoglycans were not released into the medium. The decrease in the amount of iduronic acid containing glycosaminoglycans internalized by the highly invasive cells seems to be correlated with an increased cell-associated degradation and with an apparent loss of glycosaminoglycan binding sites on the cell surface.     

Basic fibroblast growth factor is internalized through both receptor-mediated and heparan sulfate-mediated mechanisms.

Basic fibroblast growth factor (bFGF) was internalized at a rapid rate by Chinese hamster ovary (CHO) cells that do not express significant numbers of high affinity receptors for bFGF as well as CHO cells that have been transfected with cDNA encoding FGF receptor-1 or FGF receptor-2. Internalization of bFGF was completely blocked by the addition of 10 micrograms/ml heparin in the parental CHO cells but only partially inhibited in cells expressing transfected FGF receptors. Bovine aortic endothelial cells also exhibit heparin-sensitive and heparin-resistant internalization of bFGF. The internalization of bFGF through the heparin-resistant pathway in CHO cells was efficiently competed by addition of unlabeled bFGF, was proportional to the number of receptors expressed, and approached saturation, suggesting that the heparin-resistant internalization was due to high affinity receptors. Internalization of bFGF through the heparin-sensitive pathway was not efficiently competed by unlabeled bFGF and did not approach saturation at concentrations of bFGF up to 50 ng/ml, properties similar to the interaction of bFGF with low affinity heparan sulfate binding sites on the cell surface. Internalization of bFGF in CHO cells not expressing FGF receptors was inhibited by heparin, heparan sulfate, and dermatan sulfate, the same glycosaminoglycans that block binding to cell-surface heparin sulfates. Internalization of bFGF in the parental CHO cells was inhibited at the same concentrations of heparin that block binding to cell-surface heparan sulfates. Finally, inhibition of the sulfation of CHO cell heparan sulfates by the addition of chlorate or digestion of CHO cell heparan sulfates with heparinase inhibited bFGF internalization in the parental CHO cells. These results demonstrate that bFGF can be internalized through a direct interaction with cell-surface heparan sulfates. Thus, there are two pathways for internalization of bFGF: high affinity receptor-mediated and heparan sulfate-mediated.     

Binding,internalization, and degradation of antiproliferative heparan sulfate by human embryonic lung fibroblasts

Binding, internalization, and degradation of ¹²⁵I-labeled, antiproliferative, or nonantiproliferative heparan sulfate by human embryonic lung fibroblasts was investigated. Both L-iduronate-rich, antiproliferative heparan sulfate species as well as L-iduronate-poor, inactive ones were bound to trypsin-releasable, cell-surface sites. Both heparan sulfate types were bound with approximately the same affinity to one high-affinity site (K_d approximately 10⁻⁸ M) and to one (K_d approximately 10⁻⁶ M), respectively. Results of Hill-plot analysis suggested that the two sites are independent. Competition experiments with unlabeled glycosaminoglycans indicated that the binding sites had a selective specificity for sulfated, L-iduronate-rich heparan sulfate. Dermatan sulfate, which is also antiproliferative, was weakly bound to the cells. The antiproliferative effects of heparan and dermatan sulfate appeared to be additive. Hence, the two glycosaminoglycans probably exert their effect through different mechanisms. At concentrations above 5 μg/ml (approximately 10⁻⁷ M), heparan sulfate was taken up by human embryonic lung fibroblasts, suggesting that the low-affinity site represents an endocytosis receptor. The antiproliferative effect of L-iduronate-rich heparan sulfate species was also exerted at the same concentrations. The antiproliferative species was taken up to a greater degree than the inactive one, suggesting a requirement for internalization. However, competition experiments with dextran sulfate suggested that both the high-affinity and the low-affinity sites are involved in mediating the antiproliferative effect. Structural analysis of the inactive and active heparan sulphate preparations indicated that although sulphated L-iduronate appears essential for antiproliferative activity, it is not absolutely required for binding to the cells. Degradation of internalized heparan sulfate was analyzed by polyacrylamide gel electrophoresis using a sensitive detection technique. The inactive species was partially degraded, whereas the antiproliferative one was only marginally affected. J. Cell. Biochem. 64:595–604. © 1997 Wiley-Liss, Inc.     

Chondroitin sulfate and heparan sulfate-containing proteoglycans are both partners and targets of basic fibroblast growth factor-mediated proliferation in human metastatic melanoma cell lines

Basic fibroblast growth factor (FGF-2) and its respective tyrosine kinase receptors, form an autocrine loop that affects human melanoma growth and metastasis. The aim of the present study was to examine the possible participation of various glycosaminoglycans, i.e. chondroitin sulfate, dermatan sulfate and heparin on basal and FGF-2-induced growth of WM9 and M5 human metastatic melanoma cells. Exogenous glycosaminoglycans mildly inhibited WM9 cell's proliferation, which was abolished by FGF-2. Treatment with the specific inhibitor of the glycosaminoglycan sulfation, sodium chlorate, demonstrated that endogenous glycosaminoglycan/proteoglycan production is required for both basal and stimulated by FGF-2 proliferation of these cells. Heparin capably restored their growth, and unexpectedly exogenous chondroitin sulfate to WM9 and both chondroitin sulfate and dermatan sulfate to M5 cells allowed FGF-2 mitogenic stimulation. Furthermore, in WM9 cells the degradation of membrane-bound chondroitin/dermatan sulfate stimulates basal growth and even enhances FGF-2 stimulation. The specific tyrosine kinase inhibitor, genistein completely blocked the effects of FGF-2 and glycosaminoglycans on melanoma proliferation whereas the use of the neutralizing antibody for FGF-2 showed that the mitogenic effect of chondroitin sulfate involves the interaction of FGF-2 with its receptors. Both the amounts of chondroitin/dermatan/heparan sulfate and their sulfation levels differed between the cell lines and were distinctly modulated by FGF-2. In this study, we show that chondroitin/dermatan sulfate-containing proteoglycans, likely in cooperation with heparan sulfate, participate in metastatic melanoma cell FGF-2-induced mitogenic response, which represents a novel finding and establishes the central role of sulfated glycosaminoglycans on melanoma growth.     

Separation and properties of five glycosaminoglycan sulfatases from rat skin.

Chondroitin sulfates, dermatan sulfate, heparan sulfate, heparin, keratan sulfate, and oligosaccharides derived from these sulfated glycosaminoglycans have been used for the measurement of sulfatase activity of rat skin extracts. Chromatographic fractionation of the extracts followed by specificity studies demonstrated the existence of five different sulfatases, specific for 1) the nonreducing N-acetylglucosamine 6-sulfate end groups of heparin sulfate and keratan sulfate, 2) the nonreducing N-acetylgalactosamine (or galactose) 6-sulfate end groups of chondroitin sulfate (or keratan sulfate), 3) the nonreducing N-acetylgalactosamine 4-sulfate end groups of chondroitin sulfate and dermatan sulfate, 4) certain suitably located glucosamine N-sulfate groups of heparin and heparan sulfate, or 5) certain suitably located iduronate sulfate groups of heparan sulfate and dermatan sulfate. Two arylsulfatases, one of which was identical in its chromatographic behaviors with the third enzyme described above, were also demonstrated in the extracts. These results taken together with those previously obtained from studies on human fibroblast cultures suggest that normal skin fibroblasts contain at least five specific sulfatases and diminished activity of any one may result in a specific storage disease.     

The mode of action of heparan and dermatan sulfates in the regulation of hepatocyte growth factor/scatter factor.

Hepatocyte growth factor/scatter factor, in addition to binding to its specific signal-transducing receptor, Met, also interacts with both heparan and dermatan sulfates with high affinity. We have investigated the comparative role of these two glycosaminoglycans in the activation of Met by hepatocyte growth factor/scatter factor. Using glycosaminoglycan-deficient CHO pgsA-745 cells we have shown that growth factor activity is critically dependent upon glycosaminoglycans, and that heparan sulfate and dermatan sulfate are equally potent as co-receptors. Cross-linked 1:1 conjugates of growth factor and either heparan or dermatan sulfate do not dimerize under physiological conditions and are biologically active. This implies that a ternary signaling complex with Met forms in vivo. Native Met isolated from CHO pgsA-745 cells shows only very weak intrinsic affinity for heparin in vitro. Also, a heparin-derived hexasaccharide, which is the minimal size for high affinity binding to the growth factor alone, is sufficient to induce biological activity. Together these observations imply that the role of these glycosaminoglycan may be primarily to effect a conformational change in hepatocyte growth factor/scatter factor, rather than to induce a necessary growth factor dimerization, or to stabilize a ternary complex by additionally interacting with Met.     

Quantitative proteomic analysis of fibroblast nuclear proteins after stimulation with mitogen activated protein kinase inhibiting heparan sulfate

Certain structures of heparan sulfate (HS) inhibit cell proliferation of fibroblasts. Whether this inhibition is dependent on inhibition of mitogenic signaling pathways or nuclear translocation of HS is unknown. In this study we investigated possible mechanism(s) and structural requirements by which antiproliferative glycosaminoglycans exert their effects on mitogen-activated protein kinase (MAP kinase) phosphorylation, a key intermediate in cell signaling, followed by quantitative proteomic analysis of nuclear proteins by stable isotope coded affinity tags, multidimensional chromatography and tandem mass spectrometry. Serum starved human lung fibroblasts were stimulated with serum, platelet derived growth factor (PDGF-BB) or epidermal growth factor (EGF) in the presence of structurally different glycosaminoglycans. Antiproliferative heparan sulfate with a high content of 2-O-sulfated iduronic acid (IdoA-2SO4) and heavily sulfated glucosamine, and the structurally related glycosaminoglycan heparin inhibited significantly serum stimulated MAP kinase phosphorylation, by at least 80% when stimulated by serum and HS6. We hypothesized that the inhibition of the MAP kinase pathway will have effect in the nuclear proteome. Therefore an isotope coded affinity tag (ICAT) reagent labeling of nuclear proteins and tandem mass spectrometry was applied, resulting in the identification and quantification of 206 proteins. Several nuclear proteins were found to be induced or repressed due to HS stimulation, where the repression EBNA-2 co-activator and the induction of PML protein were of special interest. These results show that heparan sulfate with high content of (IdoA-2SO4) and heavily sulfated glucosamine specifically inhibits MAP kinase activation with a subsequent change in the nuclear proteome of the fibroblast.     

Cultured endothelial cells produce heparinlike inhibitor of smooth muscle cell growth

Using cultured cells from bovine and rat aortas, we have examined the possibility that endothelial cells might regulate the growth of vascular smooth muscle cells. Conditioned medium from confluent bovine aortic endothelial cells inhibited the proliferation of growth-arrested smooth muscle cells. Conditioned medium from exponential endothelial cells, and from exponential or confluent smooth muscle cells and fibroblasts, did not inhibit smooth muscle cell growth. Conditioned medium from confluent endothelial cells did not inhibit the growth of endothelial cells or fibroblasts. In addition to the apparent specificity of both the producer and target cell, the inhibitory activity was heat stable and not affected by proteases. It was sensitive flavobacterium heparinase but not to hyaluronidase or chondroitin sulfate ABC lyase. It thus appears to be a heparinlike substance. Two other lines of evidence support this conclusion. First, a crude isolate of glycosaminoglycans (TCA-soluble, ethanol-precipitable material) from endothelial cell-conditioned medium reconstituted in 20 percent serum inhibited smooth muscle cell growth; glycosaminoglycans isolated from unconditioned medium (i.e., 0.4 percent serum) had no effect on smooth muscle cell growth. No inhibition was seen if the glycosaminoglycan preparation was treated with heparinase. Second, exogenous heparin, heparin sulfate, chondroitin sulfate B (dermatan sulfate), chondroitin sulfate ABC, and hyaluronic acid were added to 20 percent serum and tested for their ability to inhibit smooth muscle cell growth. Heparin inhibited growth at concentrations as low as 10 ng/ml. Other glycosaminoglycans had no effect at doses up to 10 μg/ml. Anticoagulant and non- anticoagulant heparin were equally effective at inhibiting smooth muscle cell growth, as they were in vivo following endothelial injury (Clowes and Karnovsk. Nature (Lond.). 265:625-626, 1977; Guyton et al. Circ. Res. 46:625-634, 1980), and in vitro following exposure of smooth muscle cells to platelet extract (Hoover et al. Circ. Res. 47:578-583, 1980). We suggest that vascular endothelial cells may secrete a heparinlike substance in vivo which may regulate the growth of underlying smooth muscle cells.     

Inhibition of vascular smooth muscle cell migration by heparin-like glycosaminoglycans

Previous studies have suggested that heparin-like glycosaminoglycans may be endogenous inhibitors of smooth muscle proliferation in the vessel wall. The purpose of this study was to determine the effects of exogenous glycosaminoglycans on rat vascular (aortic) smooth muscle cell migration following wounding in vitro. Our data indicate that heparin and related molecules (iota carrageenan, dextran sulfate), but not other glycosaminoglycans (hyaluronate, chondroitin, and dermatan sulfates), inhibit smooth muscle cell motility in a cell-specific, dose-dependent, and reversible fashion. The effect of heparin was maximal (60% inhibition) at 10 μg/ml; a half-maximal effect was observed at 1 μg/ml; Heparin did not significantly affect the migration of bovine aortic endothelium or Swiss 3T3 cells. These observations support the concept that heparin-like glycosaminoglycans may be important regulators of vascular smooth muscle cell function.     

Complexing of fibronectin glycosaminoglycans and collagen

Collagen-fibronectin complexes, formed by binding of fibronectin to gelatin or collagen insolubilized on Sepharose, were found to bind 20–40% of radioactivity in [³⁵S]heparin. Fibronectin attached directly to Sepharose also bound [³⁵S]heparin, while gelatin-Sepharose without fibronectin did not. Unlabeled heparin and highly sulfated heparan sulfate efficiently inhibited the binding of [³⁵S]heparin, hyaluronic acid and dermatan sulfate were slightly inhibitory, while chondroitin sulfates and heparan sulfate with a low sulfate content did not inhibit.The interaction of heparin with fibronectin bound to gelatin resulted in complexes which required higher concentrations of urea to dissociate than complexes of fibronectin and gelatin alone. Heparin as well as highly sulfated heparan sulfate and hyaluronic acid brought about agglutination of plastic beads coated with gelatin when fibronectin was present. Neither fibronectin nor glycosaminoglycans alone agglutinated the beads.It is proposed that the multiple interactions of fibronectin, collagen and glycosaminoglycans revealed in these assays could play a role in the deposition of these substances as an insoluble extracellular matrix. Alterations of the quality or quantity of any one of these components could have important effects on cell surface interactions, including the lack of cell surface fibronectin in malignant cells.     

Heparin inhibits the attachment and growth of Balb/c-3T3 fibroblasts on collagen substrata.

In investigating the role of cell-extracellular matrix interactions in cell adhesion and growth control, the effects of heparin on cell-collagen interactions were examined. Exponentially growing Balb/c-3T3 fibroblasts were radiolabelled with 3H-thymidine and detached from tissue culture surfaces using EDTA, and cell attachment to various types of collagen substrata was assayed in the presence or absence of heparin or other glycosaminoglycans (GAGs) or dextran sulfate (40 K). Cells attached readily (70-90%) to films of types I and V, but not to type III collagen. The number of cells bound to types I and V collagen films was inhibited by 10-50% when heparin was present from 0.1-100 micrograms/ml. Cell-collagen attachment was also inhibited by dextran sulfate, and to a lesser extent by dermatan sulfate, but chondroitin sulfates A and C and hyaluronic acid showed no effect. Heparin was active even at early time points in the adhesion assay, suggesting it may disrupt cell-collagen attachment. To study the effects of heparin in modulating cell growth on collagen, growth arrested cells cultured on type I collagen films were serum stimulated in the presence of heparin or other GAGs for 3 days. Growth was inhibited (greater than 40%) only by heparin and dextran sulfate. Interaction of heparin fragments (Mr less than or equal to 6KD) with type I collagen was analyzed by affinity co-electrophoresis (Lee and Lander, 1991) and showed higher affinity heparin binding to native as compared with denatured collagen. These data suggest that sites within native collagen may mediate Balb cell-collagen and heparin-collagen interactions, and such interactions may be relevant towards understanding heparin's antiproliferative activity in vivo and in vitro.     

Specificity and affinity of binding of herpes simplex virus type 2 glycoprotein B to glycosaminoglycans.

Herpes simplex virus type 2 (HSV-2) interacts with cell surface glycosaminoglycans during virus attachment. Glycoprotein B of HSV-2 can potentially mediate the interaction between the virion and cell surface glycosaminoglycans. To determine the specificity, kinetics, and affinity of these interactions, we used plasmon resonance-based biosensor technology to measure HSV-2 glycoprotein binding to glycosaminoglycans in real time. The recombinant soluble ectodomain of HSV-2 gB (gB2) but not the soluble ectodomain of HSV-2 gD bound readily to biosensor surfaces coated with heparin. The affinity constants (Kds) were determined for gB2 (Kd = 7.7 x 10(-7) M) and for gB2 deltaTM (Kd = 9.9 x 10(-7) M), a recombinant soluble form of HSV-2 gB in which only its transmembrane domain has been deleted. gB2 binding to the heparin surface was competitively inhibited by low concentrations of heparin (50% effective dose [ED50] = 0.08 microg/ml). Heparan sulfate and dermatan sulfate glycosaminoglycans have each been suggested as cell surface receptors for HSV. Our biosensor analyses showed that both heparan sulfate and dermatan sulfate inhibited gB2 binding (ED50 = 1 to 5 microg/ml), indicating that gB2 interacts with both heparin-like and dermatan sulfate glycosaminoglycans. Chondroitin sulfate A, in contrast, inhibited gB2 binding to heparin only at high levels (ED50 = 65 microg/ml). The affinity and specificity of gB2 binding to glycosaminoglycans demonstrated in these studies support its role in the initial binding of HSV-2 to cells bearing heparan sulfate or dermatan sulfate glycosaminoglycans.     

Hypoxia modifies the effect of PDGF on glycosaminoglycan synthesis by primary human lung cells

Hypoxia, a consequence of interstitial lung diseases, may lead to secondary pulmonary hypertension and pulmonary vascular remodeling. Hypoxia induces activation and proliferation of lung cells and enhances the deposition of extracellular matrix including glycosaminoglycans (GAGs). To elucidate the cell biological mechanisms underlying the development of secondary pulmonary hypertension, we studied the effect of hypoxia on GAG synthesis by human lung cells. GAG synthesis was measured by incorporation of [(3)H]glucosamine; GAGs were isolated, purified, and characterized with GAG-degrading enzymes. Fibroblasts and vascular smooth muscle cells (VSMCs) synthesized hyaluronic acid, heparan sulfate, and chondroitin sulfates, whereas dermatan sulfate was found only in fibroblasts. Hypoxia did not influence the size or charge of the individual GAGs. However, hypoxia inhibited platelet-derived growth factor-induced [(3)H]glucosamine incorporation in secreted GAGs, especially hyaluronic acid, in VSMCs. In contrast, it stimulated GAG secretion, specifically heparan sulfate, by fibroblasts. Our results indicate that hypoxia induces modifications in GAG synthesis by human lung VSMCs and fibroblasts that may be correlated to pathophysiological manifestations in lung diseases causing hypoxia.     

The relevance of glycosaminoglycan sulfates to Apo E induced lipid uptake by hepatocyte monolayers

The binding of Apolipoprotein E supplemented triglyceride emulsions to sulfated glycosaminoglycans demonstrated specificity for the carbohydrate polymers. Glucosamine containing glycosaminoglycans with relatively less sulfate had little affinity for the Apo E emulsion whereas those with more sulfate (i.e. heparin and sulfated heparans) effectively bound the emulsion. Galactosamine containing glycosaminoglycans (chondroitin 4 sulfate and dermatan sulfate) demonstrated no binding. The Apo E induced uptake of triglyceride emulsions by hepatocytes was inhibited by highly sulfated polysaccharides (i.e. heparin, dextran sulfate) but other glycosaminoglycans which did not bind the emulsion were ineffective in this inhibition. The same sulfated compounds which inhibited the hepatocyte Apo E emulsion interaction effectively released hepatic lipase from isolated heptic perfusions. Glycosaminoglycan sulfates which did not bind the Apo E supplemented emulsions and did not inhibit hepatocyte association were ineffective in releasing lipase. A heparan mixture isolated from human liver was much less effective in inhibiting Apo E induced association of emulsions with hepatocytes, than heparin. A highly sulfated octasaccharide fraction isolated from bovine liver heparin inhibited more effectively than the human heparans but less than the heparin. Inhibition of Apo E mediated hepatocyte emulsion association was produced by a one hour exposure of the cells to either heparinase or heparanase. The heparanase was more active than the heparinase and both were effective in the presence of protease inhibitors. Enzymes hydrolyzing chondroitin sulfates and hyaluronic acid were ineffective in inhibiting the Apo E induced association. The specific binding of human low density lipoprotein to the hepatocyte was much less effected by the heparanase exposure than the Apo E mediated binding.     

Avidin is a heparin-binding protein. Affinity,specificity and structural analysis

The specificity, affinity and stoichiometry of the interaction between avidin and glycosaminoglycans (GAGs) have been investigated using heparin-coated microtiter-plate assays, a filter binding assay and surface plasmon resonance (SPR) analysis using a BIAcore 2000 biosensor. Avidin binds heparin and heparan sulfate, and chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate or hyaluronan were unable to compete for binding. Highest-affinity binding was observed with heparin, and weaker binding was seen when using heparan sulfate or low molecular weight heparin preparations. This indicated that only specific polysaccharide structures tightly interact with avidin. Approximately two avidin molecules bind to each heparin molecule with an overall affinity of 160 nM. The interaction is pH dependent, increasing five-fold upon decreasing the pH from 7.5 to 5.5, while binding was negligible at pH 9. We demonstrate the potential of fluorescent avidin derivatives as a tool for the detection of heparin and heparan sulfates on surfaces by application to both heparin immobilized on polystyrene plates and heparan sulfate on cell surfaces.     

Is there a glycosaminoglycan-related heterogeneity of the thymic epithelium?

We determined the synthesis and secretion of glycosaminoglycans by three distinct preparations of mouse cultured thymic epithelial cells. These comprised primary cultures of thymic nurse cells (TNCs), which are normally located within the cortex of the thymic lobules, as well as two murine thymic epithelial cells, bearing a mixed, yet distinct, cortico-medullary phenotype. We first identified and measured the relative proportions of the various glycosaminoglycans in the three epithelial cells. Non-sulfated glycosaminoglycans are preponderantly secreted by the TNCs, while the sulfated glycans (particularly heparan sulfate) are relatively more abundant on the cell surface. The three types of epithelial cells differ markedly in their heparan sulfate composition, mainly due to different patterns of N- and O-sulfation. In addition, the cells differ in the synthesis and secretion of other glycosaminoglycans. Thus, TNCs secrete high amounts of dermatan sulfate + chondroitin sulfate to the culture medium. IT-76M1 cells secrete high proportions of heparan sulfate while 2BH4 cells show a more equilibrated proportion of dermatan sulfate/chondroitin sulfate and heparan sulfate. The three epithelial cells also differ in their capacity to produce hyaluronic acid and 2BH4 cells are distinguished by their high rate of synthesis of this glycosaminoglycan. In conclusion, our results show that distinct thymic epithelial cells can synthesize different types of glycosaminoglycans. Although it remains to be definitely determined whether these differences reflect the in vivo situation, our data provide new clues for further understanding of how glycosaminoglycan-mediated interactions behave in the thymus.     

Divergent regulation of proteoglycan and glycosaminoglycan free chain expression in human keratinocytes and melanocytes

Summary Keratinocytes and melanocytes, which together form units of structure and function within human epidermis, are known to differ in expression of autocrine growth factors, particularly those with heparin binding affinity. Because such cytokines could be regulated by the endogenous heparinlike glycosaminoglycan, heparan sulfate, proteoglycan synthesis was compared between human keratinocytes and melanocytes cultured from a common donor. Following steady-state isotopic labeling under conditions of active growth (low density cultures) and growth inhibition (high density cultures), the sulfated polymers were isolated from conditioned media and cell extracts. We found that keratinocytes produced substantially more sulfated glycosaminoglycans than did the melanocytes. There was no evidence for hyaluronic acid synthesis by the melanocytes. The majority of [³⁵S]-sulfate labeling was in the heparan sulfates of the keratinocytes and in the chondroitin sulfates of the melanocytes. During the transition from active growth to growth inhibition, there was increased heparan sulfate proteoglycan and free chain synthesis by keratinocytes but not by melanocytes, and chondroitin sulfate proteoglycan production declined in both cell lineages. The differences may reflect divergent evolution as each cell type came to exploit those complex polysaccharides in different ways to regulate molecular pathways of growth and differentiation. The coupling of growth inhibition with augmented synthesis of heparan sulfates observed for the keratinocytes suggests a regulatory role in growth factor signaling in that cell type.