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PubMedDate not supplied

Epithelial-mesenchymal interactions in tooth morphogenesis: the roles of extracellular matrix, growth factors, and cell surface receptors.

Morphogenesis and cell differentiation in the developing tooth are controlled by a series of reciprocal interactions between the epithelial and mesenchymal tissues. The exact molecular mechanisms operating in these interactions are unknown at present, but both structural components of the extracellular matrix (ECM) and diffusible growth factors have been suggested to be involved. In this review article we summarize our findings on the distribution patterns of three ECM molecules and two cell surface receptors during tooth morphogenesis through bud, cap, and bell stages of development. The examined molecules include fibronectin, type III collagen, and tenascin, which all represent components of the mesenchymal ECM, the cell surface proteoglycan, syndecan, which functions as a receptor for interstitial matrix, and the cell surface receptor for epidermal growth factor. Based on the observed changes in distribution patterns and on experimental evidence, roles are suggested for these molecules in epithelial-mesenchymal interactions during tooth development. Fibronectin is suggested to be involved in the cell-matrix interaction that controls odontoblast differentiation. Epidermal growth factor and its receptors are suggested to be involved in a paracrine fashion in the epithelial-mesenchymal interactions regulating morphogenesis of bud- and cap-stage teeth. Tenascin and syndecan are accumulated in the dental mesenchyme during the bud stage of development, and it is suggested that they represent a couple of a cell surface receptor and its matrix ligand and that they are involved in mesenchymal cell condensation during the earliest stages of tooth morphogenesis.

Animals↗

PubMed1998

Chylomicron remnant uptake is regulated by the expression and function of heparan sulfate proteoglycan in hepatocytes.

Chylomicron remnants transport cholesterol from the intestine, and are removed from the circulation principally by the liver. While hepatic receptors, including the low density lipoprotein (LDL) receptor account for endocytosis, heparan sulfate proteoglycans (HSPG) participate in the initial binding of remnants to liver cells. To explore the interactions between HSPG and endocytosis of remnants, in the present study the expression of HSPG was inhibited in HepG2 cells transfected by a synthetic antisense oligodeoxynucleotide SYN5. Immunofluorescent staining by a monoclonal anti-syndecan antibody showed significant reduction in the expression of syndecan in SYN5-treated cells compared with control cells. Remnant binding decreased by about 50-70% in SYN5-transfected cells. Monoclonal antibodies to either heparan sulphate or the LDL receptor decreased binding by about 60-65%. The glycosylation inhibitor beta-nitrophenylxylopyranoside inhibited remnant uptake by 25%, whereas 4-nitrophenyl-beta-D-galactopyranoside had no effect on remnant binding. Heparinase completely abolished binding at appropriate concentrations. Heparitinase was less effective than hep arinase in inhibiting remnant binding. Suramin completely abolished the remnant binding. Poly-arginine, poly-lysine, and protamine all reduced remnant uptake by the cells, as did polybrene, a synthetic polycation, suggesting a role of cation-anion interactions in remnant binding. Brefeldin A, colchicine, and monensin caused the fluorescence associated with remnants to persist within the cells, confirming that blockers of tubulovesicular processes and Golgi function inhibit the intracellular transport and degradation of the remnants. Our results show that remnant binding to liver cells depends on the LDL receptor, on the expression of HSPG core proteins, and on the functionality of heparan sulfate in HSPG.

Animals↗

PubMed1997

Heparan sulfate proteoglycan expression in human lung-cancer cells.

Heparan sulfate (HS) functions as a co-factor in several signal-transduction systems that affect cellular growth, differentiation, adhesion and motility. HS, therefore, may also play a role in the malignant transformation of cells, tumor growth, cell invasiveness and the formation of tumor metastases. To explore this hypothesis, we analyzed the expression of HS and heparan sulfate proteoglycan (HSPG) in histological sections of human lung-cancer tissues and assayed for the presence of HSPGs in extracts of human lung-cancer cell lines, using a panel of native HS-, delta-HS- and HSPG (syndecan, glypican, CD44 and perlecan) core protein-specific monoclonal antibodies. Compared to normal epithelia, non-small-cell lung carcinomas, particularly poorly differentiated tumors, often expressed reduced amounts of the major cell surface-associated HSPGs (most consistently of syndecan-1). CD44 or CD44-variant proteins, in contrast, were found on all tumor cells, irrespective of their differentiation. Perlecan, a matrix-associated HSPG found in the basement membrane of normal bronchial epithelium, was consistently undetectable in invasive bronchogenic carcinomas. Staining reactions for native HS were consistently reduced in squamous-cell lung carcinomas, in the cells in contact with the stroma and in the less differentiated areas of these tumors. Reactions for delta-HS, however, were not reduced, suggesting a structural change in the HS of these tumor cells. Poorly differentiated adenocarcinomas, in contrast, yielded strong HS and delta-HS reactions. Marked differences in HSPG expression also were observed among various non-small-cell lung carcinoma cell lines. Our results suggest that poorly differentiated lung tumors have markedly altered patterns of HSPG expression, which may contribute to their invasive phenotype.

Adenocarcinoma↗

PubMed1992

Changes in histochemical distribution of cell surface heparan sulfate proteoglycan in mouse uterus during the estrous cycle and early pregnancy.

We have investigated the changes in immunolocalization of a cell surface heparan sulfate proteoglycan (HSPG) in the mouse uterus during the estrous cycle and at the time of implantation in early pregnancy. A monoclonal antibody prepared against syndecan, a cell surface HSPG from mouse mammary epithelium (gift of Dr. M. Bernfield), was reacted with unfixed and fixed frozen sections of uteri from normally cycling, 3.5 and 4.5 days pregnant, and estradiol-treated immature and ovariectomized mature mice. A polyclonal antibody prepared against basal lamina HSPG from Engelbreth-Holm-Swarm (EHS) tumor cells (gift of Dr. John Hassell) was used as a positive control. The latter showed no variation during the estrous cycle or early pregnancy. Localization of syndecan in uterine epithelium changed from basolateral to predominantly basal as the cycle progressed from metestrus toward estrus. A similar pattern was seen in immature and ovariectomized mature females that had received estradiol injections. With the onset of pregnancy, the basolateral localization became progressively less intense from 3.5 days through 4.5 days of pregnancy. Thus, cell surface HSPG distribution is modulated by hormonally dependent changes in cycling and pregnant mice, supporting previous suggestions that early pregnancy in mice is accompanied by a turnover and rearrangement of uterine epithelial cell surface.

Animals↗

PubMed1995

Chondroitin sulfate proteoglycans in the developing cerebral cortex: the distribution of neurocan distinguishes forming afferent and efferent axonal pathways.

The first thalamocortical axons to arrive in the developing cerebral cortex traverse a pathway that is separate from the adjacent intracortical pathway for early efferents, suggesting that different molecular signals guide their growth. We previously demonstrated that the intracortical pathway for thalamic axons is centered on the subplate (Bicknese et al. [1994] J. Neurosci. 14:3500-3510), which is rich in chondroitin sulfate proteoglycans (CSPGs; Sheppard et al. [1991] J. Neurosci. 11:3928-3942), whereas efferent axons cross the subplate to exit in a zone containing much less CSPG. To define the molecular composition of the subplate further, we used antibodies against CSPG core proteins and chondroitin sulfate disaccharides in an immunohistochemical analysis of their distribution in the developing neocortex of the rat. Immunolabeling for neurocan, a central nervous system-specific CSPG (Rauch et al. [1992] J. Biol. Chem. 267:19537-19547), and for chondroitin 6-sulfate and unsulfated chondroitin becomes prominent in the subplate before the arrival of thalamic afferents. Immunolabeling is initially sparse in the cortical plate but appears later in maturing cortical layers. A postnatal decline in immunolabeling occurs uniformly for most proteoglycans, but, in the somatosensory cortex, labeling for neurocan, phosphacan, and chondroitin 4- and 6-sulfate declines in the centers of the whisker barrels before the walls. In contrast to neurocan, immunolabeling for other proteoglycans is either uniformly distributed (syndecan-1, N-syndecan, 5F3, phosphacan, chondroitin 4-sulfate), restricted to axons (PGM1), distributed exclusively on nonneuronal elements (2D6, NG2, and CD44), or undetectable (9.2.27, aggrecan, decorin). Thus, neurocan is a candidate molecule for delineating the intracortical pathway of thalamocortical axons and distinguishing it from that of cortical efferents.

Afferent Pathways↗

PubMed1996

Proteoglycan gene expression in rat liver after partial hepatectomy.

Liver regeneration is regulated by several factors. Among these extracellular matrix plays a crucial role in the restoration of normal structure. Proteoglycans are major components of extracellular matrix and they are able to bind growth factors implicated in liver growth. We studied syndecan, perlecan, fibroglycan, and decorin expression after partial hepatectomy in rats by Northern blot analysis. A strong early upregulation was observed at 30 min in decorin expression which was followed by the first syndecan and perlecan peaks at 2 and 4 hours after hepatectomy. At 24 hours, which is the peak of hepatocyte DNA synthesis, all these three proteoglycans had elevated steady state transcript level. Fibroglycan message decreased after partial hepatectomy and remained low during the experimental period. The different expression pattern of the proteoglycans suggests that these extracellular matrix components may selectively influence the regeneration process.

Animals↗

PubMed1997

Altered Proteoglycan Gene Expression in Human Biliary Cirrhosis.

Proteoglycans play key roles in the physiological assembly of extracellular matrices and in the modulation of growth factor activities. During liver regeneration there is a profound remodelling of the connective tissue network with a concurrent alteration in proteoglycan gene expression. In the present study we have analyzed in detail the biochemical and molecular properties of the proteoglycans associated with biliary cirrhosis. The three major proteoglycans of human liver, namely decorin, syndecan and perlecan, were markedly elevated in the cirrhotic parenchyma as compared to normal liver tissue. Particularly elevated (eight fold) was the perlecan. This proteoglycan had not only heparan sulfate but also chondroitin and dermatan sulfate. Reverse transcriptase PCR revealed a marked enhancement of decorin and syndecan expression and detectable message for perlecan was found only in the cirrhotic liver. These results indicate that significant proteoglycan alterations are associated with the development of biliary cirrhosis and provide basis for future studies aimed at the characterization of the molecular events involved in the regulation of extracellular matrix deposition in this common human disease.

Journal Article↗

PubMed1998

Transforming growth factor-beta, basement membrane components and heparan sulphate proteoglycans in experimental hepatic schistosomiasis mansoni.

In an attempt to elucidate further the immunopathological pathways that underlie fibrogenesis induced by Schistosoma mansoni, we have studied the distribution of basement membrane compounds, heparan sulphate proteoglycans (HSPG) and the fibrogenic cytokine transforming growth factor (TGF)-beta in two models of experimental schistosomiasis mansoni (experimental murine infection and synchronous granulomas induced by injection of egg-antigen-coupled beads into the caecal vein). Deposition of the basement membrane proteins type IV collagen, laminin and entactin in schistosomal granulomas was seen 3 days after the implantation of egg-antigen-coupled beads in the liver and persisted over time (32 days). Up-regulation of the membrane-bound HSPG syndecan-1 was observed in the schistosomal granuloma. These syndecan-1-immunoreactive cells represented a distinct subpopulation of granuloma cells; they were different from both mature, unstimulated B-cells (CD40-positive) and endothelial cells (CD105-positive). Deposition of the matrix HSPG perlecan within the granuloma was most prominent 8-16 days after injection. TGF-beta expression was observed in acute (8 weeks) and chronically (13 weeks) infected mice, mainly at the periphery of the schistosomal granuloma and on Kupffer cells in the liver parenchyma. From these observations, we infer that schistosomal fibrosis is composed of various groups of matrix components and that TGF-beta, which is secreted by granuloma cells, is one of the fibrogenic mediators in schistosomal fibrogenesis.

Animals↗

PubMed1992

A cell-based assay for evaluating the interaction of heparin-like molecules and basic fibroblast growth factor.

A simple panning procedure that allows for the evaluation of interactions between various heparin-like molecules and basic FGF has been developed. This assay measures the ability of compounds to inhibit the interaction of transfected human lymphoblastoid cells, UC 729-6 (UC cells), expressing hamster syndecan and basic FGF-coated plastic plates. The transfected cells bind rapidly to basic FGF-coated plates while the control cells do not bind well. Binding of the transfected cells to basic FGF was inhibited by heparin and heparin sulfate (HS), but not by chondroitin sulfate, dermatan sulfate, keratan sulfate, and hyaluronic acid. There was little inhibition of binding by chemically modified heparin such as completely desulfated, N-acetylated heparin, completely desulfated, N-sulfated heparin, and N-desulfated, N-acetylated heparin. These results suggested that both the N-sulfate and O-sulfate groups of heparin are required for binding to basic FGF. In addition, inhibition by oligosaccharides derived from depolymerized heparin increased with fragment size; partial inhibition was observed with oligosaccharides as small as hexamers. The biochemical basis for the binding of transfected cells to basic FGF was established by showing a significant increase of 35SO4 incorporation into HS. In particular, the level of 35SO4-HS in the trypsin-releasable (cell surface) pool increased fivefold. This increase was accounted for by demonstration of the presence of HS on immunoprecipitated syndecan from the transfected cells.

Animals↗

PubMed2006

Nerve injury induces the expression of EXT2, a glycosyltransferase required for heparan sulfate synthesis.

Heparan sulfate proteoglycans, which bear long chains of heparan sulfate glycosaminoglycan, play significant roles during embryogenesis, including the formation of the CNS. However, their involvement in nerve regeneration has not yet been clarified. Here, we found that the mRNA expression of EXT2, one of the crucial enzymes for heparan sulfate-glycosaminoglycan synthesis, was markedly up-regulated in injured hypoglossal motor neurons after axotomy. In addition, immunohistochemical staining with an antibody specific for heparan sulfate-glycosaminoglycan chains demonstrated increased expression of heparan sulfate-glycosaminoglycan chains in the injured nucleus. Furthermore, the mRNA expressions of glypican-1 and syndecan-1, which are both well-known heparan sulfate proteoglycans, were prominently up-regulated in injured motor neurons. These results suggest that the biosynthesis of heparan sulfate chains promoted by EXT2 is activated in injured motor neurons, and that glypican-1 and syndecan-1 are potent candidates for heparan sulfate proteoglycans involved in peripheral nerve regeneration.

Activating Transcription Factor 3↗

PubMed2001

Biochemical characterization of integral membrane heparan sulfate proteoglycans in Sertoli cells from immature rat testis.

(35)S-Radiolabeled cultured Sertoli cells from immature rat testis were extracted with detergent and the different proteoheparan sulfate (HSPG) forms of the extract were discriminated and quantified on the basis of their high anionic charge, hydrodynamic size, lipophilic properties, susceptibility to trypsin and phosphatidylinositol phospholipase C (PI-PLC). Trypsin released 50% of total cellular HSPG corresponding to 80% of total hydrophobic HSPG. Trypsin-accessible HSPG were presumed to be integral membrane species. Trypsin-resistant HSPG, probably intracellular, distributed into non-lipophilic (37.5%) and lipophilic (12.5%) populations. Biochemical analysis of PG copurified with plasma membrane confirmed the existence of hydrophobic HSPG integrated into this structure. Among hydrophobic HSPG accessible to trypsin, 35% were PI-PLC released and radiolabeled by [(3)H]inositol indicating that about one third of integral membrane HSPG were intercalated into the plasma membrane through a phosphatidylinositol anchor (glypican type). PI-PLC-resistant forms represented HSPG inserted into the membrane through a hydrophobic segment of the core protein (syndecan type). No lipophilic PG was present in other cell compartments (culture medium, cell periphery, extracellular matrix). (125)I-Iodinated hydrophobic HSPG were deglycanated and submitted to SDS-polyacrylamide gel electrophoresis. In the glypican family, a core protein (64--65 kDa) was detected, whereas in the syndecan family, bands of 60 and 68 kDa were observed which may correspond to self-association of different core proteins. In Sertoli cell, specific functional attributes of different integral membrane HSPG forms remain to be investigated.

Animals↗

PubMed1999

Heparin, cell adhesion, and pathogenesis of inflammatory bowel disease.

Tissue repair involves a close interplay between growth factors and cell adhesion molecules. The normal healing process may be disrupted by pathophysiological states such as inflammation, due to loss of growth factors, cell adhesion molecules, or both, which results in a reduced rate of healing. Such events may occur in inflammatory bowel disease during mucosal restitution. We postulate that the beneficial response to heparin observed in inflammatory bowel disease may result from mechanisms in addition to anticoagulation. These include the restoration of high-affinity receptor binding by antiulcerogenic growth factors, such as basic fibroblast growth factor, that normally rely on the presence of heparan sulphate proteoglycans, such as syndecan-1, as co-receptors. Loss of syndecan-1 has been observed in the ulcerated mucosa of patients with inflammatory bowel disease. This loss may lead to impaired binding of basic fibroblast growth factor and a reduced rate of ulcer healing. We suggest that heparin restores high-affinity receptor binding of basic fibroblast growth, and so increases the rate of mucosal recovery.

Anticoagulants↗

PubMed1999

Proteoglycans in human malignant mesothelioma. Stimulation of their synthesis induced by epidermal, insulin and platelet-derived growth factors involves receptors with tyrosine kinase activity.

Identification of proteoglycans in two human malignant mesothelioma cell lines, one with epithelial differentiation and the other with fibroblast-like phenotype, and the effects of epidermal (EGF), insulin-like (IGF-I) and platelet-derived (PDGF-BB) growth factors on the synthesis of hyaluronan (HA) and proteoglycans (PGs) were studied. Both cell lines synthesize HA and PGs: these last were recovered both as secreted and cell-associated compounds. Chondroitin sulfate (CS) containing PGs are mainly organized as versican in the extracellular medium and as thrombomodulin and syndecan in the cell membrane. Heparan sulfate (HS) containing PGs are mainly in the form of perlecan in the culture medium, whereas cell-associated HSPGs were recovered mainly as syndecan-1, -2 and -4. Receptors for EGF, IGF-I and PDGF-BB were identified in both cell lines. In addition to cell proliferation, these growth factors stimulated the synthesis of HA and PGs, the pattern of stimulation being unique for each of them and depending on the cell phenotype. EGF increased the synthesis of HA and PGs. IGF-I showed similar stimulatory effects on the synthesis of CSPGs, whereas higher amounts were needed to influence the synthesis of HA and HSPGs, the latter only being stimulated in the epithelial cell line. PDGF-BB stimulated the synthesis of HA, HSPGs and CSPGs at low concentrations, while the stimulatory effect was abolished at higher levels. Incubation with genistein inhibited the HA and PG synthesis induced by growth factors in a mode depending on both growth factor and genistein concentrations. The results clearly suggest that the stimulatory effects of EGF, IGF-I and PDGF-BB on matrix synthesis, expressed as proteoglycan synthesis, are mediated via receptor-growth factor complexes and the protein tyrosine kinase intracellular pathway.

Chromatography, Ion Exchange↗

PubMed1997

HB-GAM (heparin-binding growth-associated molecule) and heparin-type glycans in the development and plasticity of neuron-target contacts.

HB-GAM is a secretory, extracellular matrix-associated protein that was isolated by screening for factors that enhance neurite outgrowth in rat brain neurons. The HB-GAM sequence clearly (about 50%) is homologous to that of MK (midkine) sequence, a protein discovered through screening for factors that mediate retinoic acid-induced cell differentiation. These lysine- and cysteine-rich sequences define a novel family of differentiation/growth factors, which are conserved in their structures from mammals to amphibians. HB-GAM is expressed strongly along axon pathways and target regions of axons during and prior to the stage of axonal growth in tissues. These findings, together with in vitro interactions with neurons, suggest that HB-GAM is a cell matrix-associated cue for growth cone migration. N-syndecan (syndecan-3) functions as a receptor/coreceptor in HB-GAM-induced neurite outgrowth in perinatal rat brain neurons. In addition to enhancing neurite growth in a developmentally regulated manner in early neurons, HB-GAM is accumulated at the growth cone-target interphase accompanying the onset of synaptogenesis, as evidenced by its presence at the neuromuscular junction of Xenopus and rat. In vitro studies suggest that HB-GAM functions as a local, synaptic matrix-associated factor that enhances both presynaptic and postsynaptic differentiation during development. In addition, a role in adult plasticity is suggested by studies on injury-induced and activity-dependent plasticity in rat hippocampus.

Animals↗

PubMed1997

Analysis of candidate gene co-amplification with MYCN in neuroblastoma.

Previous studies have revealed that the MYCN gene spans approximately 7kb, while the amplicon has been estimated to be 100 kb to 1 Mb long [1-3]. This implies that several other genes may be present on the MYCN amplicon. Such co-amplified genes could contribute to the malignant phenotype and might provide an explanation for why not all patients with MYCN amplification have a poor outcome. We investigated 7 neuroblastoma cell lines and 167 primary tumours for the co-amplification of candidate genes known to be present near the MYCN locus: ornithine decarboxylase, ribonucleotide reductase, syndecan-1 and a DEAD box protein gene, DDX1. We also investigated further the pG21 expressed sequence previously reported to be co-amplified with MYCN. No co-amplification with the first 3 genes was found in any of the cell lines or tumour samples. DDX1 was found to be amplified along with MYCN in 4/6 (67%) cell lines and 18/38 (47%) of the MYCN amplified tumours. No amplification of DDX1, ODC1, RRM2 or syndecan-1 was found in the absence of MYCN amplification. DDX1 co-amplification was observed to occur mainly in stage 4 or 4S patients. With the exclusion of those with 4S disease, patients with DDX1 co-amplification had a significantly shorter mean (+/- SE) disease-free interval (4.1 +/- 1.4, n = 8 months) compared with patients with MYCN amplification alone (19.6 +/- 4.5, n = 17) (P = 0.04, Welch's unpaired t-test). The pG21 sequence was identical to part of the DDX1 gene. These observations indicate that there is at least 1 other gene co-amplified with MYCN in a proportion of cases and that those patients with DDX1 co-amplification tend to relapse more quickly. It also implies that the MYCN amplicon is of varied size and/or position relative to the MYCN gene.

Adolescent↗

PubMed2006

Modulation of cell-fibronectin matrix interactions during tissue repair.

Environmental signals from the extracellular matrix (ECM) are transmitted by cell surface receptors that connect to the actin cytoskeleton and to multiple intracellular signaling pathways. To dissect how the ECM regulates cell functions, we are using a three-dimensional (3D) fibrin-fibronectin matrix, resembling the wound provisional matrix. Fibroblasts adhere to fibronectin in this matrix via concomitant engagement of alpha 5 beta 1 integrin receptors and syndecan-4, a transmembrane proteoglycan. An adhesive phenotype is developed with actin stress fibers and activation of focal adhesion kinase (FAK) and Rho GTPase. Lack of syndecan-4 engagement, as occurs in the presence of the ECM protein tenascin-C, promotes a motile phenotype; FAK and Rho signaling are downregulated and filopodia are extended. Fibronectin matrices have distinct effects on two other receptors: alpha 4 beta 1 and beta v beta 3 integrins. Although alpha 4 beta 1 does not naturally support strong cell interactions with a fibrin-fibronectin matrix, binding is dramatically enhanced by proteolytic cleavage of fibronectin. Conversely, activity of alpha v beta 3 is stimulated by multimeric fibronectin fibrils showing that the organization of fibronectin differentially affects integrin functions. Thus, deposition of additional ECM components, expression of co-receptors for ECM, cleavage of adhesive proteins, and the architecture of the ECM microenvironment are different mechanisms for modulating cell responses to fibronectin matrix.

Animals↗

PubMed1995

Expression of cell-surface proteoglycan mRNA by human articular chondrocytes.

The expression of six cell-surface proteoglycans (syndecan, fibroglycan, amphiglycan, glypican, betaglycan and CD44) was studied at the mRNA level. Analysis was performed by Northern blotting using total RNA preparations from freshly isolated articular chondrocytes obtained from both juveniles and adults. Similar results were obtained for both age groups. By far the most abundant message was that for amphiglycan, CD44 message was next in relative abundance, and the messages for fibroglycan, glypican and betaglycan were all expressed at low levels. Syndecan message could not be detected by this technique. This pattern of expression was different to that observed in cultured skin fibroblasts, where the messages for amphiglycan, CD44, fibroglycan and glypican were all expressed at a similar level. In contrast with the fibroblasts, where the amphiglycan message exhibits no size polymorphism, the chondrocyte amphiglycan message is present in three polymorphic forms, due to the use of alternative polyadenylation signals. When the newly isolated chondrocytes are maintained in monolayer culture for several passages, the amphiglycan message heterogeneity reverts to that characteristic of the fibroblasts. Thus human articular chondrocytes are characterized by both their high level of amphiglycan message expression and their use of alternative polyadenylation signals.

Adult↗

PubMed1995

Proteoglycans in polarized epithelial Madin-Darby canine kidney cells.

Madin-Darby canine kidney (MDCK) cells were cultured on polycarbonate filters to study the synthesis and sorting of proteoglycans in polarized epithelial cells. Two strains of MDCK cells were used. MDCK I cells resemble distal tubule epithelial cells, and MDCK II cells share some characteristics with proximal tubule cells. Both strains were grown to confluency and labelled with [35S]sulphate for 24 h. The apical and basolateral media and the cell fractions were harvested and analysed by DEAE ion-exchange chromatography. A large portion of the [35S]sulphate-labelled macromolecules bound strongly to the ion-exchange columns, and could be eluted in three distinct peaks. The latest eluting peak was demonstrated to contain almost exclusively chondroitin sulphate, whereas peak 2 contained mostly heparan sulphate, demonstrated by using chondroitinase ABC and nitrous acid (pH 1.5) respectively to depolymerize the [35S]glycosaminoglycan chains. Peak 1 contained negligible amounts of proteoglycans. Large differences could be observed in proteoglycan sorting in MDCK I and II cells. Strain I secreted approx. 67% of the proteoglycans to the apical side and 17% to the basolateral side. The cell fraction contained 17% of the proteoglycans after 24 h of labelling. In contrast, 19% of the proteoglycans were sorted to the apical side of MDCK II cells and 61% to the basolateral side, whereas the cell fraction contained 20%. Furthermore, the level of [35S]proteoglycan biosynthesis (apical and basolateral media and cell fraction total) was higher in MDCK I cells than in strain II. Based on the amount of material degraded by chondroitinase ABC and nitrous acid respectively, and the total amounts of [35S]proteoglycans recovered from the cells, it was calculated that the MDCK I strain synthesized approx. 56% chondroitin sulphate and 44% heparan sulphate. In contrast, the MDCK II strain synthesized 69% heparan sulphate and 31% chondroitin sulphate. To further identify the [35S]proteoglycans synthesized by MDCK I and II cells, antibodies against perlecan, versican and syndecan were used. The antibody against mouse syndecan did not cross-react with any of the proteoglycans produced in MDCK I or II cells. Both MDCK I and II cells expressed perlecan; 57-61% could be recovered from the basolateral fractions and 18-34% from the apical medium. Versican was also found in both MDCK I and II cells. Compared with perlecan, a larger percentage of versican (43-53%) was found in the cell fractions.

Animals↗