Large Proteoglycan Research Articles

Structure and Regulation of the Versican Promoter: THE VERSICAN PROMOTER IS REGULATED BY AP-1 AND TCF TRANSCRIPTION FACTORS IN INVASIVE HUMAN MELANOMA CELLS

Versican is a large chondroitin sulfate proteoglycan of the extracellular matrix that is involved in a variety of cellular processes. We showed previously that versican, which is overexpressed in cutaneous melanomas as well as in premalignant lesions, contributes to melanoma progression, favoring the detachment of cells and the metastatic dissemination. Here, we investigated the transcriptional regulation of the versican promoter in melanoma cell lines with different levels of biological aggressiveness and stages of differentiation. We show that versican promoter up-regulation accounts for the differential expression levels of mRNA and protein detected in the invasive SK-mel-131 human melanoma cells. The activity of the versican promoter increased 5-fold in these cells in comparison with that measured in non-invasive MeWo melanoma cells. Several transcriptional regulatory elements were identified in the proximal promoter, including AP-1, Sp1, AP-2, and two TCF-4 sites. We show that promoter activation is mediated by the ERK/MAPK and JNK signaling pathways acting on the AP-1 site, suggesting that BRAF mutation present in SK-mel-131 cells impinge upon the up-regulation of the versican gene through signaling elicited by the ERK/MAPK pathway. This is the first time the AP-1 transcription factor family has been shown to be related to the regulation of versican expression. Furthermore, deletion of the TCF-4 binding sites caused a 60% decrease in the promoter activity in SK-mel-131 cells. These results showing that AP-1 and TCF-4 binding sites are the main regulatory regions directing versican production provide new insights into versican promoter regulation during melanoma progression.

Ligation of TLR2 by Versican: A Link Between Inflammation and Metastasis

Versican, a large extracellular matrix proteoglycan, accumulates both in tumor stroma and cancer cells. It participates in cell adhesion, migration, and angiogenesis, all features of invasion and metastasis. However, the mechanism(s) whereby versican promotes cancer invasion and metastasis is not yet fully understood. A recent study has documented that versican can activate tumor-infiltrating myeloid cells through toll-like receptor (TLR) 2 and its co-receptors TLR6 and CD14 and elicit the production of proinflammatory cytokines including TNF-alpha that enhance tumor metastasis. As both resident fibroblasts and endothelial cells (ECs) also express functional TLR2 and its co-receptors, we hypothesized that, in addition to myeloid cells, versican may trigger the activation of both fibroblasts and ECs. Of interest, TLR2-mediated activation of EC and fibroblast has been observed to increase the secretion of interleukin-8, a proinflammatory CXC chemokine that potentiates neutrophil infiltration and angiogenesis, as well as metastatic growth. Ligation of TLR2 by versican appears to be directly involved in the activation of multiple types of cells in tumor stroma and the induction of inflammatory cytokine secretion, providing a link between inflammation and cancer metastasis. Accordingly, antagonists of versican and TLR2 restrain the activation of tumor stromal cells, which may offer a novel approach to cancer therapy by targeting tumor microenvironment.

A Human Folliculoid Microsphere Assay for Exploring Epithelial– Mesenchymal Interactions in the Human Hair Follicle

The search for more effective drugs for the management of common hair growth disorders remains a top priority, both for clinical dermatology and industry. In this pilot study, we report a pragmatic organotypic assay for basic and applied hair research. The patented technique produces microdroplets, which generate human folliculoid microspheres (HFMs), consisting of human dermal papilla fibroblasts and outer root sheath keratinocytes within an extracellular matrix that simulates elements of the hair follicle mesenchyme. Studying a number of different markers (for example, proliferation, apoptosis, cytokeratin-6, versican), we show that these HFMs, cultured under well-defined conditions, retain several essential epithelial-mesenchymal interactions characteristic for human scalp hair follicle. Selected, recognized hair growth-modulatory agents modulate these parameters in a manner that suggests that HFMs allow the standardized preclinical assessment of test agents on relevant human hair growth markers under substantially simplified in vitro conditions that approximate the in vivo situation. Furthermore, we show by immunohistochemistry, reverse transcriptase-PCR, and DNA microarray techniques that HFMs also offer a useful discovery tool for the identification of target genes and their products for candidate hair drugs. HFM thus represent an instructive modern experimental and screening tool for basic and applied hair research in the human system.

Versican/PG-M Assembles Hyaluronan into Extracellular Matrix and Inhibits CD44-mediated Signaling toward Premature Senescence in Embryonic Fibroblasts

Versican/PG-M is a large chondroitin sulfate proteoglycan of the extracellular matrix which interacts with hyaluronan at the N-terminal G1 domain, composed of A, B, and B' subdomains. Recently, we generated knock-in mice Cspg2(Delta3/Delta3), whose versican, without the A subdomain, has decreased hyaluronan (HA) binding affinity, thereby exhibiting reduced deposition of versican in the extracellular matrix. Here, we show that the Cspg2(Delta3/Delta3) fibroblasts within 20 passages proliferate more slowly and acquire senescence. Whereas the extracellular matrix of the wild type fibroblasts exhibited a network structure of hyaluronan and versican, that of the Cspg2(Delta3/Delta3) fibroblasts exhibited approximately 35 and approximately 85% deposition of versican and HA, without such a structure. The Cspg2(Delta3/Delta3) fibroblasts showed a substantial increase of ERK1/2 phosphorylation and expression of senescence markers p53, p21, and p16. Treatment of wild type fibroblasts with hyaluronidase and exogenous hyaluronan enhanced ERK1/2 phosphorylation, and treatment with an anti-CD44 antibody that blocks HA-CD44 interaction inhibited the phosphorylation. These results demonstrate that versican is essential for matrix assembly involving hyaluronan and that diminished versican deposition increases free hyaluronan fragments that interact with CD44 and increase phosphorylation of ERK1/2, leading to cellular senescence.

Stimulation of Small Proteoglycan Synthesis by the Hyaluronan Synthesis Inhibitor 4-Methylumbelliferone in Human Skin Fibroblasts

Human skin fibroblasts cultured with 4-methylumbelliferone (MU), a hyaluronan synthesis inhibitor, produce a hyaluronan-deficient extracellular matrix (See []). Our present study investigated the effects of MU on proteoglycan, which is the other main component of the extracellular matrix, and interacts with hyaluronan. Proteoglycans isolated from culture medium in the presence or absence of MU were characterized by gel-filtration chromatography, ion-exchange HPLC, electrophoresis, and immunoblotting. We found that MU had only a negligible effect on the synthesis of large proteoglycan but increased the production of small proteoglycan in comparison with cultures lacking MU. This small proteoglycan was identified by immunoblotting as decorin. The structures of decorin synthesized in the presence and absence of MU were compared by gel-filtration chromatography, and the data indicated that cells incubated with MU produced a larger decorin molecule than cells incubated without MU. Furthermore, the two decorins had galactosaminoglycan chains of different sizes. These results suggest that MU inhibits the synthesis of hyaluronan and accelerates production of the larger decorin in the extracellular matrix.

Serum Amyloid A, but Not C-Reactive Protein, Stimulates Vascular Proteoglycan Synthesis in a Pro-Atherogenic Manner

Inflammatory markers serum amyloid A (SAA) and C-reactive protein (CRP) are predictive of cardiac disease and are proposed to play causal roles in the development of atherosclerosis, in which the retention of lipoproteins by vascular wall proteoglycans is critical. The purpose of this study was to determine whether SAA and/or CRP alters vascular proteoglycan synthesis and lipoprotein retention in a pro-atherogenic manner. Vascular smooth muscle cells were stimulated with either SAA or CRP (1 to 100 mg/L) and proteoglycans were then isolated and characterized. SAA, but not CRP, increased proteoglycan sulfate incorporation by 50 to 100% in a dose-dependent manner (P < 0.0001), increased glycosaminoglycan chain length, and increased low-density lipoprotein (LDL) binding affinity (K(d), 29 microg/ml LDL versus 90 microg/ml LDL for SAA versus control proteoglycans; P < 0.005). Furthermore, SAA up-regulated biglycan via the induction of endogenous transforming growth factor (TGF)-beta. To determine whether SAA stimulated proteoglycan synthesis in vivo, ApoE(-/-) mice were injected with an adenovirus expressing human SAA-1, a null virus, or saline. Mice that received adenovirus expressing SAA had increased TGF-beta concentrations in plasma and increased aortic biglycan content compared with mice that received either null virus or saline. Thus, SAA alters vascular proteoglycans in a pro-atherogenic manner via the stimulation of TGF-beta and may play a causal role in the development of atherosclerosis.

Ultrastructural organisation of arthritis corneal stroma

Abstract Purpose Normal corneal stroma consists of uniformly organised collagen fibrils regulated by corneal proteoglycans. The focus of the study is the ultrastructural organisations of the collagen fibrils and proteoglycans in patients suffering with corneal melting due to rheumatoid arthritis (RA). Methods Corneas of two patients (ages 50 &amp; 61 years) suffering from RA were fixed in 2.5% glutaraldehyde containing cuprolinic blue in sodium acetate buffer. The tissue were dehydrated in a graded series of ethanol and embedded and polymerised in spurs resin. The sections were studied under an electron microscope. Results The epithelial cells were degenerated and Bowman’s layer was replaced by fibrous pannus containing large abnormal proteoglycans and lucent spaces. In the stroma, longitudinally running collagen fibrils were curled and fused to each other. In cross section, collagen fibrils were not rounded and were separated with large spacings. Large proteoglycans and lucent spaces were present throughout stroma. The Descemet’s membrane was followed by posterior collagenous layer. Degenerated keratocytes were present in the pannus and throughout the stroma. Conclusion The collagen fibrils and proteoglycans were severely degenerated which resulted in the disorganisation of the corneal stroma. We believed that due to the disease, alteration in keratan sulphate and chondroitin sulphate might occur which possibly affected the structure of the corneal proteoglycans (lumican, keratocan and mimican) which were responsible for the collagen fibrils organisation.

Myofibroblast and smooth muscle actin in scarred corneal stroma

Abstract Purpose Mohan et al, (2003) hypothesised that after corneal injury, the site of injury is invaded by myofibroblasts (MFBCs) derived from dividing keratocytes. We report here the distribution of myofibroblasts (MFBCs), and cellular location of α‐‐ smooth muscle actin (α‐‐SMA) in scarred cornea. Methods Three penetrating keratoplasty buttons, two with post‐zoster scarring and one with hypertrophic dendriform epitheliopathy, were fixed in: 1) 2.5% glutaraldehyde containing cuprolinic blue for ultrastructural studies and 2) 4% paraformaldehyde for immunogold localisation. Primary α‐‐SMA and vimentin antibodies were localised by gold conjugates. Results Large numbers of MFBCs were observed in the sub‐epithelial collagenous pannus, posterior stroma and posterior collagenous layer in these corneas. The MFBCs were very large in size, lay in layers and occasionally fused with each other. Labelling for α‐SMA was very strong in all MFBCs. Labelling of α‐SMA was also observed in basal epithelial cells. MFBCs contained very prominent rough endoplasmic reticulum, vimentin filaments and large nuclei. Significantly, large proteoglycans (nm) and collagen fibrils (nm) were present around these cells. Conclusion It has been suggested that cytokynes released from the overlying epithelium act to maintain the myofibroblast phenotype (Jester et al 1999a, 1999b). We hypothesise that MFBCs synthesise large amounts of proteoglycan which disrupt the organisation of collagen fibrils and lead to the opacities of scarred cornea. The finding of labelling for α‐SMA in basal epithelial cells suggests that some epithelial cells may transform into MFBCs.

Formation of stromal collagen fibrils and proteoglycans in the developing zebrafish cornea

Collagen fibrils and proteoglycans are the main components of the corneal extracellular matrix and corneal transparency depends crucially on their proper arrangement. In the present study, we investigated the formation of collagen fibrils and proteoglycans in the developing cornea of the zebrafish, a model organism used to study vertebrate embryonic development and genetic disease. We employed thin-section electron microscopy to investigate the ultrastructure of the zebrafish cornea at different developmental stages. The layering of the zebrafish cornea into an epithelium, a Bowman's layer, stroma and endothelium was observed starting at 72 hr post-fertilization. At this stage, the stroma contained orthogonally arranged collagen fibrils and small proteoglycans. The density of proteoglycans increased gradually throughout subsequent development of the cornea. In the stroma of 2-week-old larvae, the collagen fibrils were organized into thin lamellae and were separated by very large, randomly distributed proteoglycans. At 4 weeks, a regular arrangement of proteoglycans in relation to the collagen fibrils was observed for the first time and the lamellae were also thickened. The present study, for the first time, provides ultrastructural details of collagen fibril and proteoglycan development in the zebrafish cornea. Furthermore, it directly correlates the collagen fibril and proteoglycan composition of the zebrafish cornea with that of the human cornea. The similarities between the two species suggest that the zebrafish could serve as a model for investigating the genetics of human corneal development and diseases.

On the association between amyloid fibrils and glycosaminoglycans; possible interactive role of Ca2+ and amyloid P-component.

We have previously reported the specific association of glycosaminoglycans (GAG) and proteoglycans (PG) with amyloid fibrils and characterized the polysaccharides directly extracted from amyloid-laden tissues. In the present study we further elucidate the association between purified amyloid fibrils and GAG/PG with special reference to those GAG/PG associated with amyloid P-component (AP) and the interactive role of Ca2+ ions. Amyloid fibrils were isolated from human hepatic AA amyloid employing water extraction with and without preceding removal of AP, an extrafibrillar protein component of all amyloids, using sodium citrate. GAG/PG co-isolated with the amyloid extracts, with and without AP, were isolated and characterized. Agarose-affinity chromatography of extracts containing AP was performed, and the GAG associated with this extrafibrillary protein were characterized as well. Several different GAG/PG populations were demonstrated in the various extracts. The abolition of calcium-dependent binding markedly influenced the amount of GAG/PG recovered in the fibril extracts, as well as the total amount of amyloid material obtained. Thus, it seems that calcium plays an important role in the association between the fibrils and the sugar moieties, and that a significant fraction of the GAG found in amyloid exhibits a Ca(2+)-dependent fibril-GAG interaction. No significant difference in the proportion between galactosaminoglycans and glucosamines was, however, disclosed when the two extraction protocols were compared, suggesting that no particular GAG species has a higher affinity for the fibrils themselves. Both dermatan/chondroitin sulphate and heparan sulphate identified in the present study exhibited a Ca(2+)-dependent interaction with AP, supporting previous findings. However, the amyloid-associated galactosaminoglycans found, especially the large PG appearing in small amounts, seemed to have a higher affinity for the extrafibrillar AP than the other GAG.

Effects of glucosamine on proteoglycan loss by tendon, ligament and joint capsule explant cultures

To investigate the effect of glucosamine on the loss of newly synthesized radiolabeled large and small proteoglycans by bovine tendon, ligament and joint capsule. The kinetics of loss of (35)S-labeled large and small proteoglycans from explant cultures of tendon, ligament and joint capsule treated with 10mM glucosamine was investigated over a 10-day culture period. The kinetics of loss of (35)S-labeled small proteoglycans and the formation of free [(35)S]sulfate were determined for the last 10 days of a 15-day culture period. The proteoglycan core proteins were analyzed by gel electrophoresis followed by fluorography. The metabolism of tendon, ligament and joint capsule explants exposed to 10mM glucosamine was evaluated by incorporation of [(3)H]serine and [(35)S]sulfate into protein and glycosaminoglycans, respectively. Glucosamine at 10mM stimulated the loss of small proteoglycans from ligament explant cultures. This was due to the increased loss of both macromolecular and free [(35)S]sulfate to the medium indicating that glucosamine affected the release of small proteoglycans as well as their intracellular degradation. The degradation pattern of small proteoglycans in ligament was not affected by glucosamine. In contrast, glucosamine did not have an effect on the loss of large or small proteoglycans from tendon and joint capsule or large proteoglycans from ligament explant cultures. The metabolism of cells in tendon, ligament and joint capsule was not impaired by the presence of 10mM glucosamine. Glucosamine stimulated the loss of small proteoglycans from ligament but did not have an effect on small proteoglycan catabolism in joint capsule and tendon or large proteoglycan catabolism in ligament, tendon or synovial capsule. The consequences of glucosamine therapy at clinically relevant concentrations on proteoglycan catabolism in joint fibrous connective tissues need to be further assessed in an animal model.

Intracellular Versican Expression in Mesenchymal Spindle Cell Tumors Contrasts With Extracellular Expression in Epithelial and Other Tumors—A Tissue Microarray-based Study

Versican is a large chondroitin sulfate proteoglycan that is an integral component of the extracellular matrix protein. It regulates cell proliferation, adhesion, and migration, and is expressed in a variety of normal tissues and tumors. We studied the pattern of versican expression in various epithelial, mesenchymal, neural, and hematopoietic tumors using immunohistochemistry on tissue microarrays. The primary antibody used was mouse monoclonal antibody to versican (clone 8S270, 1:4000, US Biological). Sections from 3 healing wounds were also included to demonstrate versican expression in reactive tissues. The extracellular matrix in all tissues including all tumors (epithelial and nonepithelial) was positive for versican. However, intracellular cytoplasmic expression of versican was seen only in spindle cells, for example, fibroblasts in healing wounds, 11 of 16 (69%) gastrointestinal stromal tumors and 12 of 42 (28%) smooth muscle tumors. Intracellular versican was not seen in any other tumor [0/344 carcinomas (64 breast, 63 prostate, 61 colorectal, 59 lung, 68 ovarian, and 29 thyroid), 0/22 glioblastoma multiforme, 0/46 lymphomas, and 0/21 melanomas]. As versican plays a role in cell proliferation, differentiation, adhesion, and migration, its differential expression in spindle cell tumors may be associated with the differentiation, progression, and spread of these tumors, which is different from epithelial tumors.

Interaction of Syntenin-1 and the NG2 Proteoglycan in Migratory Oligodendrocyte Precursor Cells

Migration of oligodendrocyte precursors along axons is a necessary prerequisite for myelination, but little is known about underlying mechanisms. NG2 is a large membrane proteoglycan implicated in oligodendrocyte migration. Here we show that a PDZ domain protein termed syntenin-1 interacts with NG2 and that syntenin-1 is necessary for normal rates of migration. The association of syntenin-1 with NG2, identified in a yeast two-hybrid screen, was confirmed by colocalization of both proteins within processes of oligodendroglial precursor cells and by coimmunoprecipitation from cell extracts. Syntenin-1 also colocalizes with NG2 in "co-capping" assays, demonstrating a lateral association of both proteins in live oligodendrocytes. RNA interference-mediated down-regulation of syntenin-1 in glial cells results in a significant reduction of migration in vitro, as does the presence of polyclonal antibody against NG2. Thus syntenin plays a role in the migration of oligodendroglial precursors, and we suggest that NG2-syntenin-1 interactions contribute to this.

The regulation of aggrecanase ADAMTS-4 expression in human Achilles tendon and tendon-derived cells

Several members of the ADAMTS (A Disintegrin And Metalloproteinase with ThromboSpondin motifs) family have been identified as aggrecanases, whose substrates include versican, the principal large proteoglycan in the tendon extracellular matrix. We have characterized the expression of ADAMTS-4 in human Achilles tendon and tendon-derived cells. ADAMTS-4 mRNA levels were higher in ruptured tendon compared with normal tendon or chronic painful tendinopathy. In tissue extracts probed by Western blotting, mature ADAMTS-4 (68 kDa) was detected only in ruptured tendons, while processed ADAMTS-4 (53 kDa) was detected also in chronic painful tendinopathy and in normal tendon. In cultured Achilles tendon cells, transforming growth factor-β (TGF-β) stimulated ADAMTS-4 mRNA expression (typically 20-fold after 24 h), while interleukin-1 induced a smaller, shorter-term stimulation which synergised markedly with that induced by TGF-β. Increased levels of immunoreactive proteins consistent with mature and processed forms of ADAMTS-4 were detected in TGF-β-stimulated cells. ADAMTS-4 mRNA was expressed at higher levels by tendon cells in collagen gels than in monolayer cultures. In contrast, the expression of ADAMTS-1 and -5 mRNA was lower in collagen gels compared with monolayers, and these mRNA showed smaller or opposite responses to growth factors and cytokines compared with that of ADAMTS-4 mRNA. We conclude that both ADAMTS-4 mRNA and ADAMTS-4 protein processing may be differentially regulated in normal and damaged tendons and that both the matrix environment and growth factors such as TGF-β are potentially important factors controlling ADAMTS aggrecanase activities in tendon pathology.

Cartilage is held together by elastic glycan strings. Physiological and pathological implications

Animal shapes are maintained by connective tissue extracellular matrices (ECMs). ECM shapes depend on keeping collagen fibrils in the right places, held by regular frequent proteoglycan (PG) bridges attached at specific sites. The PGs carry anionic glycosaminoglycan (AGAG) 'strings' that span and determine interfibrillar distances, thus holding us together. I called these repeating structures 'shape modules'. The strings are aggregated antiparallel chains of dermochondan, keratan and chondroitan sulphates (DS, KS and CS); stabilised by hydrophobic and H-bonds. Shape modules are elastic. AGAG/AGAG interactions break under stress and reform when the stress is removed and/or they contain an elastic sugar, L-iduronate (in DS). Cartilage ECMs are also based on shape modules. Depots therein of aggrecan, the large PG which carries many chains of CS and KS, imbibe water, thereby exerting swelling pressure. External pressure forces this water into the elastic shape modules, from whence it is returned post compression. Cartilage anisotropic responses (along and at right angles to shape module axes) to compressive and tensile stresses are now interpretable. Degradation of shape modules in osteoarthrosis reduces these responses. Inability to hold collagen fibrils together results in imbibition of excess water, fissuring and erosion, characteristic of this condition.

Regulation of hyaluronan and versican deposition by growth factors in fibrosarcoma cell lines

Versican, a large chondroitin sulphate proteoglycan and hyaluronan (HA), a non-sulphated glycosaminoglycan are major constituents of the pericellular matrix. In many neoplastic tissues, changes in the expression of versican and HA affect tumour progression. Here, we analyse the synthesis of versican and hyaluronan by fibrosarcoma cells, and document how the latter is affected by PDGF-BB, bFGF and TGFB2, growth factors endogenously produced by these cells. Fibrosarcoma cell lines B6FS and HT1080 were utilised and compared with normal lung fibroblasts (DLF). The major versican isoforms expressed by DLF and B6FS cells were V0 and V1. Treatment of B6FS cells with TGFB2 showed a significant increase of V0 and V1 mRNAs. Versican expression in HT1080 cells was not significantly affected by any of the growth factors. In addition, TGFB2 treatment increased versican protein in DLF cells. HA, showed approximately a 2-fold and a 9-fold higher production in DLF cells compared to B6FS and HT1080 cells, respectively. In HT1080 cells, HA biosynthesis was significantly increased by bFGF, whereas, in B6FS cells it was increased by TGFB2 and PDGF-BB. Furthermore, analysis of HA synthases (HAS) expression indicated that HT1080 expressed similar levels of all three HAS isoforms in the following order: HAS2> HAS3> HAS1. bFGF shifted that balance by increasing the abundance of HAS1. The major HAS isoform expressed by B6FS cells was HAS2. PDGF-BB and TGFB2 showed the most prominent effects by increasing both HAS2 and HAS1 isoforms. In conclusion, these growth factors modulated, through upregulation of specific HAS isoforms, HA synthesis, secretion and net deposition to the pericellular matrix.

Structure and function of basement membrane proteoglycans.

Basement membranes contain at least three different proteoglycans. These are a large, low buoyant density heparan sulphate proteoglycan and two smaller, high density proteoglycans with either heparan sulphate or chondroitin sulphate side-chains. The large (Mr 400K-600K and small (Mr 130K) heparan sulphate proteoglycans were purified from the mouse EHS tumour. These proteoglycans are immunologically related by sharing some protein core antigenic determinants (epitopes) but do not cross-react with cell-surface heparan sulphate proteoglycans or with proteoglycans from interstitial connective tissue. This indicates that they belong to a distinct family of proteoglycans. Structural models were developed, based on electron microscopy and analytical ultracentrifugation, demonstrating that the small proteoglycan contains on average four heparan sulphate chains of about 30 nm in length, while the large proteoglycan consists of three long (about 90 nm) heparan sulphate chains connected to one end of a large core protein. Single heparan sulphate chains were isolated from the EHS tumour proteoglycans and from the corresponding proteoglycans from Reichert's membrane of the mouse embryo. The heparan sulphate from Reichert's membrane bound to antithrombin with high affinity and was found to contain the unique 3-O-sulphated glucosamine residue previously identified in the antithrombin-binding region of heparin. The EHS tumour heparan sulphate showed a higher N-/O-sulphate ratio and a lower affinity for antithrombin.

Cartilage proteoglycans.

The structure of the protein core of the high molecular weight aggregating proteoglycan from pig laryngeal cartilage has been investigated. Mild trypsin digestion of proteoglycan aggregates released a large (Mr approximately equal to 150K) protein-rich fragment that contained the hyaluronate-binding region (Mr 66K). Rotary-shadowing electron microscopy of this preparation showed it to contain 'double globe' structures, similar to those seen with intact proteoglycans. Interaction studies and immunochemical evidence showed that one of the globular domains was the binding region. The second globular domain did not interact with hyaluronate or share any major antigenic determinants with the binding region and its function remains unknown. Further evidence from rotary shadowing also suggested that the protein core contained a third globular domain at the C-terminal end. The complete protein core sequence thus contains long folded globular protein regions, in addition to the extended regions bearing glycosaminoglycan chains. Studies of proteoglycan turnover in explants of pig articular cartilage showed that proteoglycan fragments were continuously released into the medium during culture. These included large non-aggregating proteoglycan fragments, free binding region and also link protein. Proteoglycans retained within the cartilage matrix remained intact and able to aggregate. Only in the presence of interleukin 1 was there evidence of more extensive proteolytic digestion. The results suggest normal turnover to be a conservative mechanism involving the selective cleavage of proteoglycan close to the hyaluronate-binding region. This releases the major glycosaminoglycan-bearing domain and enables it to diffuse out of the matrix. The site of the initial cleavage appears to be in the region of the N-terminal globular domains.

Effect of osteogenic protein‐1 on the matrix metabolism of bovine tendon cells

Tendon rupture is a common sports injury in adults. However, the mechanical properties of repair tissue are inferior to those of normal tissue. To accelerate tendon healing, an in vivo approach using growth factors has been applied and has shown evidence for the efficacy of biological stimulation of the repair process. Recombinant human osteogenic protein-1 (rhOP-1) has been shown to be effective in stimulating matrix production by various connective tissues. To test the effect of rhOP-1 on the matrix metabolism of tendon cells in vitro, bovine tendon cells were cultured in monolayer with various doses of rhOP-1 for 7 days. The addition of rhOP-1 to cell culture media resulted in significant increases in cell proliferation, DNA content, and the synthesis of proteoglycans (PGs) and collagen, compared to control cultures. The relative percentage of large PGs in the OP-1 culture was higher than that in the control culture. In conclusion, we show for the first time that rhOP-1 stimulates the proliferation of tendon cells and their ability to synthesize and accumulate PGs and collagen in their extracellular matrix. These biological properties may be used in the tissue-engineering of tendon tissues.

Comparison of the expression of agrin, a basement membrane heparan sulfate proteoglycan, in cholangiocarcinoma and hepatocellular carcinoma

Heparan sulfate proteoglycans mediate cell adhesion and control the activities of numerous growth and motility factors. They play a critical role in carcinogenesis and tumor progression. Agrin is a large multidomain heparan sulfate proteoglycan associated with basement membranes in several tissues. The expression of agrin in the liver has recently been described under physiologic and pathologic conditions. However, little is known about its role in malignancies. We aimed to study the mRNA and protein expression of agrin in cholangiocarcinoma (CC) and focused on the differences between CC and hepatocellular carcinoma (HCC). Eighty surgically removed liver specimens were studied by immunohistochemistry. Representative samples were used for immunoblotting. mRNA expression was measured in 32 samples by real-time polymerase chain reaction. By immunohistochemistry, agrin was seen around bile ducts and blood vessels within the portal areas in the normal liver. Although no expression was found within the hepatic lobules, agrin was deposited in the neovascular basement membrane in HCCs. Agrin was abundant in the tumor-specific basement membrane in well-differentiated areas of CCs, whereas with immunostaining, it was fragmented, decreased, or it even disappeared in less differentiated areas and sites of infiltration. By real-time polymerase chain reaction, up-regulation of agrin expression was measured in HCCs compared with that in the normal liver. CC samples showed an even higher expression of agrin. Immunoblotting confirmed these findings. Our results indicate that agrin might play an important role in neoangiogenesis in human HCC, being a part of the newly formed vasculature. In CC, however, agrin might be involved in tumor progression.