Vascular endothelial growth factor stimulates chemotactic migration of primary human osteoblasts.

Recent studies have indicated a critical role for vascular endothelial growth factor (VEGF) during the process of endochondral ossification, in particular in coupling cartilage resorption with bone formation. Therefore, we studied the chemoattractive and proliferative properties of human VEGF-A on primary human osteoblasts (PHO) and compared these data with the effects of human basic fibroblast growth factor (bFGF) and human bone morphogenetic protein-2 (BMP-2). Furthermore, initial experiments were carried out to characterize VEGF-binding proteins on osteoblastic cells possibly involved in the response. For the first time, to our knowledge, we could demonstrate a chemoattractive effect of VEGF-A, but not VEGF-E, on primary human osteoblasts. The effect of VEGF-A was dose-dependent and did not reach a maximum within the concentration range tested (up to 10 ng/mL). The maximal effect observed was a chemotactic index (CI) of 2 at a concentration of 10 ng/mL. bFGF and BMP-2 exhibited maxima at 1.0 ng/mL with CI values of 2.5 and 2, respectively. In addition to its effect on cell migration, VEGF-A stimulated cell proliferation by up to 70%. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed the expression of VEGF receptors VEGFR-1 (Flt-1), VEGFR-2 (Kdr), and VEGFR-3 (Flt-4), as well as neuropilin-1 and -2. An in vitro kinase assay failed to demonstrate activation of VEGFR-2 upon stimulation with either VEGF-E or VEGF-A, consistent with the idea that the effect of VEGF-A on primary human osteoblasts is mediated via VEGFR-1. Taken together, our data establish that human osteoblasts respond to VEGF-A, suggesting a functional role for this growth factor in bone formation and remodeling.

Different usage of the glycosaminoglycan attachment sites of biglycan.

Biglycan is a member of the small leucine-rich proteoglycan family. Its core protein comprises two chondroitin/dermatan sulfate attachment sites on serine 42 and serine 47, respectively, which are the fifth and tenth amino acid residues, respectively, after removal of the prepro peptide. Because the regulation of glycosaminoglycan chain assembly is not fully understood and because of the in vivo existence of monoglycanated biglycan, mutant core proteins were stably expressed in human 293 and Chinese hamster ovary cells in which i) either one or both serine residues were converted into alanine or threonine residues, ii) the number of acidic amino acids N-terminal of the respective serine residues was altered, and iii) a hexapeptide was inserted between the mutated site 1 and the unaltered site 2. Labeling experiments with [(35)S]sulfate and [(35)S]methionine indicated that serine 42 was almost fully used as the glycosaminoglycan attachment site regardless of whether site 2 was available or not for chain assembly. In contrast, substitution of site 2 was greatly influenced by the presence or absence of serine 42, although additional mutations demonstrated a direct influence of the amino acid sequence between the two sites. When site 2 was not substituted with a glycosaminoglycan chain, there was also no assembly of the linkage region. These results indicate that xylosyltransferase is the rate-limiting enzyme in glycosaminoglycan chain assembly and implicate a cooperative effect on the xylosyl transfer to site 2 by xylosylation of site 1, which probably becomes manifest before the removal of the propeptide. It is shown additionally that biglycan expressed in 293 cells may still contain the propeptide sequence and may carry heparan sulfate chains as well as sulfated N-linked oligosaccharides.

Influence of decorin expression on transforming growth factor-beta-mediated collagen gel retraction and biglycan induction.

Complex formation of transforming growth factor-beta (TGF-beta) with the small proteoglycan decorin has been considered to inactivate the cytokine. However, neither the TGF-beta-mediated stimulation of the retraction of collagen lattices in culture nor the enhanced transcription of biglycan were influenced by an excess of native decorin in the culture medium. In contrast, when MG-63 osteosarcoma cells were transfected with sense- or antisense-decorin-cDNA, which led to an over- or under-expression of the proteoglycan, they responded to TGF-beta differently. An inverse correlation between decorin expression and the TGF-beta-mediated stimulation of collagen gel retraction and biglycan induction, respectively, was found. These results are best explained by assuming that decorin is not inactivating but sequestering TGF-beta in the extracellular matrix.

Extracellular matrix and cytokines: a functional unit.

The extracellular matrix (ECM) as well as soluble mediators like cytokines can influence the behavior of cells in very distinct as well as cooperative ways. One group of ECM molecules which shows an especially broad cooperativity with cytokines and growth factors are the proteoglycans. Proteoglycans can interact with their core proteins as well as their glycosaminoglycan chains with cytokines. These interactions can modify the binding of cytokines to their cell surface receptors or they can lead to the storage of the soluble factors in the matrix. Proteoglycans themselves may even have cytokine activity. In this review we describe different proteoglycans and their interactions and relationships with cytokines and we discuss in more detail the extracellular regulation of the activity of transforming growth factor-beta (TGF-beta) by proteoglycans and other ECM molecules. In the third part the interaction of heparan sulfate chains with fibroblast growth factor-2 (FGF-2, basic FGF) as a prototype example for the interaction of heparin-binding cytokines with heparan sulfate proteoglycans is presented to illustrate the different levels of mutual dependence of the cytokine network and the ECM.

Lipoprotein lipase-mediated interactions of small proteoglycans and low-density lipoproteins.

According to numerous studies low-density lipoproteins (LDL) are supposed to interact with the glycosaminoglycan chain(s) of proteoglycans, e.g. with decorin and biglycan, which themselves are subject to receptor-mediated endocytosis. We tested, therefore, whether complexes of LDL and small proteoglycans can be endocytosed by either the LDL- or the small proteoglycan uptake mechanism. However, neither was the endocytosis of LDL significantly influenced by proteoglycans nor that of proteoglycans by LDL. This negative result could be explained by the observation that in vitro complex formation takes place only in buffers of low ionic strength. Under physiological conditions additional molecules may be necessary for complex stabilization. Lipoprotein lipase (LpL) which binds LDL was also able to interact with high affinity with decorin and its glycosaminoglycan-free core protein, both interactions being heparin-sensitive. Regardless of the presence or absence of LDL, LpL stimulated the endocytosis of decorin 1.5-fold, whereas LpL mediated a 4-fold stimulation of LDL uptake in the absence of decorin. No significant additional effect was seen in the presence of small concentrations of proteoglycans whereas in the presence of 1 microM decorin the endocytosis of [125I]LDL was reduced in normal as well as in LDL receptor-deficient fibroblasts. These observations could best be explained by assuming that LpL/LDL complexes are internalized upon binding to membrane-associated heparan sulphate and that small proteoglycans interfere with this process. It could not be ruled out, however, that a small proportion of the complexes is also taken up by the small proteoglycan receptor.

Alternative exon usage of rat septins.

Septins represent a family of phylogenetically conserved proteins required for cytokinesis. Their presence in pre- and postsynaptic neuronal membranes suggests a general function as scaffolds for membrane reorganization. The transcriptional regulation of all septins examined so far is complex, resulting in alternatively spliced variants. We focus here on the rat homologue of the gene for the human septin MSF, a truncated form of which, designated eseptin, had been described previously. It will be shown here that there is an alternative usage of the first exon by two forms, named exon r1a and r1b, respectively. Exon r1a, but not exon r1b, contains a part of the coding sequence while the start of translation for the remaining coding sequence resides in the second exon. The complete genomic organization was resolved and data on the temporal and spatial expression of this septins are presented.

[Does low intensity, pulsed ultrasound speed healing of scaphoid fractures?]. / Beschleunigt niedrig intensiver, gepulster Ultraschall die Heilung von Skaphoidfrakturen?

Since pulsed low-intensity ultrasound (frequency: 1.5 MHz, pulsed by 1 kHz, signal burst width: 200 microseconds, intensity: 30 mW/cm2) has been proven to stimulate fracture healing both clinically and experimentally, our question was whether this therapy also accelerates healing of fresh stable scaphoid fractures. Addressing this question, we did the following prospective randomized clinical trial. Regarding the results of former clinical fresh fracture studies by Heckman and Kristiansen, we postulated that low intensity ultrasound accelerates healing by about 30%. Based on this thesis, we calculated that 30 patients divided into two groups would be necessary to show significant differences between the standard treatment (treated by casting) and an adjunctive ultrasound treatment (treated by casting and additional daily 20 minutes ultrasound treatment) if present. Diagnosis and healing was assessed by CT scans every two weeks. CT's were analyzed by two independent radiologists and one hand surgeon. Furthermore, areas of cancellous bone bridging in correlation to the diameter of the scaphoid was measured in each CT scan. The results showed ultrasounded fractures healing in 43.2 +/- 10.9 days versus 62 +/- 19.2 days in the control group (p < 0.01). Trabecular bridging six weeks after injury showed 81.2% +/- 10.4% healed in the ultrasound-stimulated fractures versus 54.6% +/- 29% in the control (p < 0.05). Our study results confirm those of Heckman and Kristiansen and show a similar acceleration of bone healing. Low intensity ultrasound is successful in accelerating the healing of fresh scaphoid fractures.

Interaction of thrombospondin-1 and heparan sulfate from endothelial cells. Structural requirements of heparan sulfate.

Cell surface-associated heparan sulfate proteoglycans, predominantly perlecan, are involved in the process of binding and endocytosis of thrombospondin-1 (TSP-1) by vascular endothelial cells. To investigate the structural properties of heparan sulfate (HS) side chains that mediate this interaction, the proteoglycans were isolated from porcine endothelial cells and HS chains obtained thereof by beta-elimination. To characterize the structural composition of the HS chains and to identify the TSP-1-binding sequences, HS was disintegrated by specific chemical and enzymatic treatments. Cell layer-derived HS chains revealed the typical structural heterogeneity with domains of non-contiguously arranged highly sulfated disaccharides separated by extended sequences containing predominantly N-acetylated sequences of low sulfation. Affinity chromatography on immobilized TSP-1 demonstrated that nearly all intact HS chains possessed binding affinity, whereas after heparinase III treatment only a small proportion of oligosaccharides were bound with similar affinity to the column. Size fractioning of the bound and unbound oligosaccharides revealed that only a specific portion of deca- to tetradecasaccharides possessed TSP-1-binding affinity. The binding fraction contained over 40% di- and trisulfated disaccharide units and was enriched in the content of the trisulfated 2-O-sulfated L-iduronic acid-N-sulfated-6-O-sulfated glucosamine disaccharide unit. Comparison with the disaccharide composition of the intact HS chains and competition experiments with modified heparin species indicated the specific importance of N- and 6-O-sulfated glucosamine residues for binding. Further depolymerization of the binding oligosaccharides revealed that the glucosamine residues within the TSP-1-binding sequences are not continuously N-sulfated. The present findings implicate specific structural properties for the HS domain involved in TSP-1 binding and indicate that they are distinct from the binding sequence described for basic fibroblast growth factor, another HS ligand and a potential antagonist of TSP-1.

Decorin endocytosis: structural features of heparin and heparan sulphate oligosaccharides interfering with receptor binding and endocytosis.

Receptor-mediated endocytosis of decorin depends on its core-protein-mediated interaction with a 51 kDa membrane protein, which, in addition to its core-protein-binding site, carries a binding site for glycosaminoglycan chains. Membrane-associated heparan sulphate as well as heparin are known to have an inhibitory effect on decorin endocytosis by cultured skin fibroblasts. In this study, structural features of both glycosaminoglycans required for binding to the 51 kDa protein and for inhibiting decorin endocytosis, were investigated. Upon digestion of [(3)H]glucosamine-labelled heparan sulphate with heparinase III, dodeca- and higher saccharides were able to interact with the receptor protein. In comparison with unbound fragments of the same size, bound fragments were enriched in N-sulphated disaccharides carrying one or two sulphate ester groups. Using heparinase III-generated fragments from [(35)S]sulphate-labelled heparan sulphate chains, binding of fragments as small as octasaccharides could be detected. Competition experiments between dermatan sulphate and chemically modified heparin revealed that N- and 6-O-sulphation of glucosamine residues are important structural elements for binding to the receptor, whereas iduronate-2-O-sulphate groups contribute to binding only to a limited extent. However, with respect to the inhibition of decorin endocytosis, 2-O-desulphation had a quantitatively similar effect to 6-O-desulphation. Furthermore, for maximal inhibition of decorin endocytosis, longer fragments were required than for binding to the receptor. Thus, it appears that heparin/heparan sulphate has to interact with additional component(s) for effective inhibition of decorin uptake.

Decorin, biglycan and their endocytosis receptor in rat renal cortex.

BACKGROUND: Among the small proteoglycans, biglycan and decorin have been proposed to be potent modulators of TGF-beta-mediated inflammatory kidney diseases. They were considered to become induced during glomerulonephritis and to subsequently inactivate the cytokine. METHODS: Decorin and biglycan as well as their endocytosis receptor were investigated in normal rat renal cortex, in anti-Thy-1 glomerulonephritis, in polycystic kidneys, in the remnant kidney following 5/6-nephrectomy, and in kidneys from the Milan normotensive strain by immunohistochemistry and in situ hybridization. Northern blots were used for the detection of mRNA expression for decorin and biglycan in isolated glomeruli. Functional aspects of the endocytosis of decorin and biglycan were studied in cultured mesangial cells. RESULTS: In the normal adult rat kidney decorin was expressed preferentially by Bowman's capsule and by interstitial connective tissue cells, but only in trace amounts by mesangial cells. In contrast, biglycan was found in tubular epithelial cells, in association with glomerular capillaries, podocytes and occasionally in the mesangium. In the tubulointerstitium of diseased kidneys (polycystic kidneys, 5/6-nephrectomy, kidneys from the Milan normotensive strain) there was a general up-regulation of decorin expression, while biglycan was localized only in distinct foci of fibrotic lesions. Glomerulosclerosis (5/6-nephrectomy, Milan normotensive strain) was associated with an increased staining for both decorin and biglycan within glomeruli. However, even in the anti-Thy-1 model of an acute mesangioproliferative glomerulonephritis where the greatest accumulation of decorin was found there was only a slight enhancement of decorin mRNA in isolated glomeruli. Decorin and biglycan become degraded upon receptor-mediated endocytosis. Immunohistochemical investigations indicated that the pattern of expression of the receptor protein correlated well with the immunolocalization of both decorin and biglycan. In vitro experiments with cultured mesangial cells provided direct evidence for the expression of the receptor and for the cell's capability to endocytose decorin as well as biglycan. CONCLUSIONS: Decorin and biglycan are characterized by a distinct expression pattern in the normal rat kidney, whereas the presence of their endocytosis receptor protein correlates with the expression of both proteoglycans. Decorin is almost completely absent in the normal mesangium. Both proteoglycans become up-regulated in various models of renal disease. The mesangial accumulation of decorin in the anti-Thy-1 glomerulonephritis that is observed in spite of the only slightly enhanced mRNA expression could result from decreased decorin turnover and/or increased mesangial retention.

Receptor-mediated endocytosis of decorin: involvement of leucine-rich repeat structures.

Decorin, a small dermatan sulfate proteoglycan, is characterized by a core protein with central leucine-rich repeat structures and a single glycosaminoglycan chain. It is catabolized by receptor-mediated uptake and subsequent intralysosomal degradation. In the present study, the localization of the receptor binding site(s) along the core protein was investigated. Various recombinant decorin fragments were consistently able to inhibit the endocytosis of wild-type decorin. The most potent inhibitory peptides were those which encompassed the central Leu125-Val230 region, i.e., the fifth to eighth leucine-rich repeat, or at least a part of it. The peptide Leu125-Val230 bound directly to the 51-kDa endocytosis receptor, and Fab fragments of antibodies against this peptide inhibited the endocytosis of decorin in a dose-dependent manner. Decorin constructs expressed in human 293 cells and comprising the full-length coding region or lacking sequences N- and/or C-terminally of the Leu125-Val230 region were all endocytosed with similar clearance rates. These data suggest that the N- and C-terminal domains of the core protein are not required for endocytosis. The receptor binding site is rather represented by contiguous leucine-rich repeat structures of the central part of the core protein. This conclusion is supported by competition experiments with biglycan, a structurally related small proteoglycan.

Chondroitin/dermatan sulphate promotes the survival of neurons from rat embryonic neocortex.

Recently we have shown that biglycan, a small chondroitin sulphate proteoglycan of the extracellular matrix, supports the survival of cultured neurons from the developing neocortex of embryonic day 15 rats. Here we investigate the structure-function relationship of this neurotrophic proteoglycan and show that chondroitin/dermatan sulphate chains are the active moieties supporting survival. Heparin, a highly sulphated glucosaminoglycan, is less active than the galactosaminoglycans (chondroitin-4-sulphate, chondroitin-6-sulphate and dermatan sulphate), whereas hyaluronic acid, an unsulphated glucosaminoglycan, does not support neuron survival. Galactosaminoglycans must be in direct contact with neurons to cause survival. Experiments with elevated potassium concentrations and antagonists of voltage-gated calcium channels exclude the involvement of membrane depolarization. However, genistein and an erbstatin analogue, which are inhibitors of tyrosine kinases with low specificity, abolished neuron survival in the presence of chondroitin/dermatan sulphate, whereas a selective inhibitor of neurotrophin receptor kinases (K252a) had no suppressive effect. Thus, yet unidentified tyrosine kinases are involved in the chondroitin/dermatan sulphate-dependent survival of neocortical neurons. In the embryonic stages of rat neocortical development chondroitin sulphate is mainly located in layers I, V and VI and the subplate. Chondroitin sulphate expression is maintained after birth, extends up to cortical layer IV on postnatal day 7, and is down-regulated until postnatal day 21 concomitant with the period of naturally occurring cell death. The latter observation is consistent with a putative role of chondroitin sulphate in the control of neuron survival during cortical histogenesis.

Isolation and cellular localization of the decorin endocytosis receptor.

The small dermatan sulfate proteoglycans decorin and biglycan are efficiently internalized by a variety of cells of mesenchymal origin. This process is modulated, at least under tissue culture conditions, by cell surface-associated heparan sulfate proteoglycans. Receptor proteins of 51 and 26 kDa, respectively, bind to leucine-rich repeat structures of the core proteins of the small proteoglycans but also to highly sulfated domains of heparan sulfate. The 51 kDa protein was purified from rat brain tissue by subcellular fractionation, heparin affinity chromatography and subsequent SDS-PAGE, and was used for raising a polyclonal antiserum. Affinity-purified antibodies also recognize the 26 kDa protein and a few other low molecular weight proteins, suggesting that these proteins represent proteolytic degradation products of the 51 kDa receptor. By confocal laser microscopy, it could be demonstrated that the affinity-purified antibody reacted at 0 degree C with a protein that became internalized and was transported to a perinuclear compartment during 15 min of incubation at 37 degrees C. These findings provide direct evidence that the receptor protein(s) are internalized together with the ligand and reach an endosomal compartment where further sorting can occur.

Decorin-type I collagen interaction. Presence of separate core protein-binding domains.

Interactions between the core protein of the small dermatan sulfate proteoglycan decorin and type I collagen have been considered to influence the kinetics of collagen fibrillogenesis and the diameter of and the distance between the fibrils. A variety of recombinant core protein fragments were expressed in Escherichia coli, extracted from inclusion bodies, and renatured in the presence of bovine serum albumin, which was essential for obtaining functional activity. A recombinant protein lacking the first 14 amino acids of the mature core protein (P15-329) interacted with reconstituted type I collagen fibrils and inhibited collagen fibrillogenesis almost as efficiently as intact decorin purified from fibroblast secretions under non-denaturing conditions. Peptides comprising amino acids 15-183 (P15-183) and 185-329 (P185-329) were able to compete for the binding of wild-type decorin, with P15-183 being more active than P185-329. Several other peptides were much less effective. Binding studies using radioactively labeled peptides P15-183 and P185-329 gave direct evidence for the independent binding of both peptides. Peptides 15-183 and 15-125 had the capability of inhibiting collagen fibrillogenesis, whereas peptide 185-329 was inactive. These data suggest (i) that there are at least two separate binding domains for the interaction between decorin core protein and type I collagen and (ii) that binding is not necessarily correlated with an alteration of collagen fibrillogenesis.

Endocytosis of decorin by bovine aortic endothelial cells. off.

The small dermatan sulfate proteoglycan decorin is efficiently internalized by a variety of cells of mesenchymal origin. Decorin-binding receptor proteins of 51 and 26 kDa are involved in this process. Uptake is modulated by highly sulfated heparan sulfate which interacts with the receptor proteins, too. Compared with cultured skin fibroblasts, bovine aortic endothelial cells have a lower capacity for decorin endocytosis whereas their apparent concentration of receptor proteins is even higher. The low internalization rate is attributed to the greater occupancy of receptor proteins by heparan sulfate of the plasma membrane and/or the extracellular matrix. Growth of endothelial cells on Falcon 3090 tissue culture inserts made possible to study decorin uptake from the apical and basolateral membrane, respectively. Decorin uptake was at the limit of detection when the proteoglycan was added to the basolateral compartment. Uptake via the apical membrane was at least as efficient as in monolayer cultures on plastic. The basolateral membrane, however, was enriched in receptor proteins, but also in heparan sulfate proteoglycans. It is, therefore, suggested that endothelial cells are especially involved in the clearance of decorin which is present in blood plasma.

Selective inactivity of TGF-beta/decorin complexes.

Previous studies had shown that binding of TGF-beta to the small proteoglycan decorin results in its inactivation. Indeed, in osteosarcoma cells the addition of decorin prevented the TGF-beta 1-mediated up-regulation of biglycan synthesis. However, the down-regulation of proteoglycan-100 remained unaltered. Even in the presence of a 100,000-fold molar excess of decorin, TGF-beta 1 was fully active in U937 monocytes with respect to the inhibition of cell proliferation. There was no inhibition of the TGF-beta-mediated stimulation of the retraction of fibroblast-populated collagen lattices. Thus, the formation of TGF-beta/decorin complexes leads to the neutralization of distinct effects only.

Biosynthesis and interactions of small chondroitin/dermatan sulphate proteoglycans.

This review pays special attention to the structure and functions of two chondroitin/dermatan sulphate proteoglycans which are members of the family of small leucine-rich proteoglycans of the extracellular matrix. Novel data are presented indicating the importance of the core protein for the determination of the extent of glycosaminoglycan modification. Decorin as well as biglycan are able to associate specifically with type I collagen fibrils and to interact with several other components of the extracellular matrix. Recombinant fragments of both proteoglycans inhibit collagen fibrillogenesis. Evidence is presented for the functional diversity of decorin core protein. Considering the proposal of a glycosaminoglycan-glycosaminoglycan interaction, data are presented indicating that exclusively glycosaminoglycan chains containing a peptide moiety of more than two amino acids are able to interact with native decorin, suggesting that protein-protein or protein-glycosaminoglycan interactions are of importance in this respect, too. The interactions of decorin with growth factors are discussed, and it is shown that complexes of transforming growth factor-beta and decorin are still able to exhibit some but not all the specific effects of the uncomplexed cytokine.

Small proteoglycans.

In this review the structure and functions of two non-related proteoglycan families are discussed. One family represents a group of extracellular matrix macromolecules characterized by core proteins with leucine-rich repeat motifs. Within this family special attention is given to those members which carry chondroitin or dermatan sulfate glycosaminoglycan chains. The second family is characterized by repeat sequences of serine and glycine. Their members are products of a single core protein gene and are characteristic constituents of secondary vesicles in cells of the haematopoietic lineage.

Different galactosaminoglycan composition of small proteoglycans from osteosarcoma cells.

The expression of the core proteins and the co-polymeric structure of the glycosaminoglycan chains of three different small proteoglycans (biglycan, decorin, proteoglycan-100) have been examined in the human osteosarcoma cell line MG-63. The three proteoglycans, which are carrying either one or two chondroitin/dermatan sulphate chains, were synthesized in a similar molar ratio, as determined by [35S]methionine as well as by [35S]sulphate incorporation. After sulphate ester formation, they were secreted into the culture medium with similar kinetics. Immune staining with monospecific antibodies revealed that at least biglycan and proteoglycan-100 were present in all individual cells. However, in contrast to these similarities, the glycosaminoglycan moiety of proteoglycan-100 was composed exclusively of chondroitin 4- and 6-sulphate repeating units, whereas biglycan and decorin contained hybrid polymers of chondroitin and dermatan sulphate with approximately 90% 4-sulphated disaccharide repeating units. Treatment with transforming growth factor-beta resulted in a marked down-regulation of proteoglycan-100 synthesis without significant alteration of its glycosaminoglycan structure. Up-regulation of biglycan and moderate down-regulation of decorin were accompanied by a small decrease in the conversion of chondroitin to dermatan sulphate disaccharide units in both cases. The specific stimulation of the biosynthesis of proteoglycan-100 by tumour necrosis factor-alpha was without consequence for its glycosaminoglycan composition. Treatment with tumour necrosis factor-alpha had no influence on the synthesis and glycosaminoglycan structure of biglycan and decorin. These findings support the proposal of the importance of the core protein for the determination of the extent of glycosaminoglycan modification.

Influence of membrane-associated heparan sulfate on the internalization of the small proteoglycan decorin.

The small dermatan sulfate proteoglycan decorin is efficiently internalized by a variety of cells of mesenchymal origin. Previous studies had implicated the involvement of 51- and 26-kDa receptor proteins in this uptake process. The surface localization of these proteins has now been demonstrated by labeling with a membrane-impermeant, biotinylating reagent. The human keratinocyte cell line HaCaT exhibited only about 5% of the clearance rate of fibroblasts for exogeneously added decorin, although it was not deficient in the 51- and 26-kDa proteins. Evidence is presented that plasma membrane-associated heparan sulfate influences receptor trafficking and contributes to the low internalization rate of the receptors in keratinocytes: (i) Heparitinase digestion of intact keratinocytes led to an approximately 10-fold increase in the efficiency of decorin endocytosis. (ii) Endocytosis of decorin was increased more than 10-fold in keratinocytes in the presence of protamine, a cationic, heparan sulfate-binding protein. This effect is considered to be caused by competition between protamine and the endocytosis receptor for cell surface-associated heparan sulfate. (iii) Preincubation of keratinocytes with heparan sulfate-degrading enzymes at 37 degrees C led to a decrease of receptor proteins localized at the cell surface as judged by subsequent surface labeling at 0 degree C. (iv) An alteration of the biosynthesis of heparan sulfate proteoglycans by p-nitrophenyl-beta-xyloside was accompanied by an increased yield of intracellularly located receptor proteins. Plasma membrane-associated heparan sulfate from keratinocytes differed from the corresponding species of fibroblasts in quantity and quality. It is, therefore, suggested that the intracellular trafficking of the decorin receptor proteins is influenced by the amount and/or the composition of membrane-associated heparan sulfate.