Cloned mouse mast cells resemble, by ultrastructure, immature mast cells observed in vivo. These mast cell clones can be grown in the absence of any other cells, facilitating direct investigations of their biochemistry and function. We find that cloned mast cells express plasma membrane receptors (Fc epsilon R) that bind mouse IgE with an equilibrium constant (KA) similar to that of normal mouse peritoneal mast cells. In addition, cloned mast cells do not display detectable la antigens and cannot enhance lg secretion when added to lymphocyte cultures or mediate natural killer lysis. In the presence of 1 mM sodium butyrate, cloned mast cells stop dividing and acquire abundant electron-dense cytoplasmic granules similar to those of mature mast cells. Their histamine content increases concomitant with cytoplasmic granule maturation and may exceed that of untreated mast cells by 50-fold. Unlike peritoneal mast cells, cloned mast cells incorporate 35SO4 into chondroitin sulfates rather than heparin. These findings demonstrate that, unlike fully differentiated mouse peritoneal mast cells, cloned immature mouse mast cells contain no heparin and low levels of histamine. In addition, they establish that high-affinity Fc epsilon R are expressed early in mast cell maturation, well before completion of cytoplasmic granule synthesis and mediator storage.

BACKGROUND

Human central nervous system-stem cells grown as neurospheres (hCNS-SCns) self-renew, are multipotent, and have potential therapeutic applications following trauma to the spinal cord. We have previously shown locomotor recovery in immunodeficient mice that received a moderate contusion spinal cord injury (SCI) and hCNS-SCns transplantation 9 days post-injury (dpi). Engrafted hCNS-SCns exhibited terminal differentiation to myelinating oligodendrocytes and synapse-forming neurons. Further, selective ablation of human cells using Diphtheria toxin (DT) abolished locomotor recovery in this paradigm, suggesting integration of human cells within the mouse host as a possible mechanism for the locomotor improvement. However, the hypothesis that hCNS-SCns could alter the host microenvironment as an additional or alternative mechanism of recovery remained unexplored; we tested that hypothesis in the present study.

RESULTS

Stereological quantification of human cells using a human-specific cytoplasmic marker demonstrated successful cell engraftment, survival, migration and limited proliferation in all hCNS-SCns transplanted animals. DT administration at 16 weeks post-transplant ablated 80.5% of hCNS-SCns. Stereological quantification for lesion volume, tissue sparing, descending serotonergic host fiber sprouting, chondroitin sulfate proteoglycan deposition, glial scarring, and angiogenesis demonstrated no evidence of host modification within the mouse spinal cord as a result of hCNS-SCns transplantation. Biochemical analyses supplemented stereological data supporting the absence of neural stem-cell mediated host repair. However, linear regression analysis of the number of engrafted hCNS-SCns vs. the number of errors on a horizontal ladder beam task revealed a strong correlation between these variables (r = -0.78, p<0.05), suggesting that survival and engraftment were directly related to a quantitative measure of recovery.

CONCLUSIONS

Altogether, the data suggest that the locomotor improvements associated with hCNS-SCns transplantation were not due to modifications within the host microenvironment, supporting the hypothesis that human cell integration within the host circuitry mediates functional recovery following a 9 day delayed transplant.

NRP1 (neuropilin-1) is a co-receptor for members of the VEGF (vascular endothelial growth factor) family in endothelial cells, but is increasingly implicated in signalling induced by other growth factors. NRP1 is expressed in VSMCs (vascular smooth muscle cells), but its function and the mechanisms involved are poorly understood. The present study aimed to determine the role of NRP1 in the migratory response of HCASMCs (human coronary artery smooth muscle cells) to PDGF (platelet-derived growth factor), and to identify the signalling mechanisms involved. NRP1 is highly expressed in HAoSMCs (human aortic smooth muscle cells) and HCASMCs, and modified in VSMCs by CS (chondroitin sulfate)-rich O-linked glycosylation at Ser612. HCASMC migration induced by PDGF-BB and PDGF-AA was inhibited by NRP1 siRNA (small interfering RNA), and by adenoviral overexpression of an NRP1 mutant lacking the intracellular domain (Ad.NRP1ΔC). NRP1 co-immunoprecipitated with PDGFRα (PDGF receptor α), and immunofluorescent staining indicated that NRP1 and PDGFRα co-localized in VSMCs. NRP1 siRNA also inhibited PDGF-induced PDGFRα activation. NRP1-specific siRNA, Ad.NRP1ΔC and removal of CS glycans using chondroitinase all inhibited PDGF-BB and -AA stimulation of tyrosine phosphorylation of the adapter protein, p130Cas (Cas is Crk-associated substrate), with little effect on other major signalling pathways, and p130Cas knockdown inhibited HCASMC migration. Chemotaxis and p130Cas phosphorylation induced by PDGF were inhibited by chondroitinase, and, additionally, adenoviral expression of a non-glycosylatable NRP1S612A mutant inhibited chemotaxis, but not p130Cas phosphorylation. These results indicate a role for NRP1 and NRP1 glycosylation in mediating PDGF-induced VSMC migration, possibly by acting as a co-receptor for PDGFRα and via selective mobilization of a novel p130Cas tyrosine phosphorylation pathway.

Proteoglycans are expressed in various tissues on cell surfaces and in the extracellular matrix and display substantial heterogeneity of both protein and carbohydrate constituents. The functions of individual proteoglycans of the nervous system are not well characterized, partly because specific reagents which would permit their isolation are missing. We report here that the monoclonal antibody 473HD, which binds to the surface of early differentiation stages of murine astrocytes and oligodendrocytes, reacts with the chondroitin sulfate/dermatan sulfate hybrid epitope DSD-1 expressed on a central nervous system chondroitin sulfate proteoglycan designated DSD-1-PG. When purified from detergent-free postnatal days 7 to 14 mouse brain extracts, DSD-1-PG displays an apparent molecular mass between 800-1,000 kD with a prominent core glycoprotein of 350-400 kD. Polyclonal anti-DSD-1-PG antibodies and monoclonal antibody 473HD react with the same molecular species as shown by immunocytochemistry and sequential immunoprecipitation performed on postnatal mouse cerebellar cultures, suggesting that the DSD-1 epitope is restricted to one proteoglycan. DSD-1-PG promotes neurite outgrowth of embryonic day 14 mesencephalic and embryonic day 18 hippocampal neurons from rat, a process which can be blocked by monoclonal antibody 473HD and by enzymatic removal of the DSD-1-epitope. These results show that the hybrid glycosaminoglycan structure DSD-1 supports the morphological differentiation of central nervous system neurons.

Plasmodium falciparum-infected erythrocytes bind to chondroitin sulfate A (CSA) in the placenta via the VAR2CSA protein, a member of the P. falciparum erythrocyte membrane protein-1 family, leading to life-threatening malaria in pregnant women with severe effects on their fetuses and newborns. Here we describe the structure of the CSA binding DBL3x domain, a Duffy binding-like (DBL) domain of VAR2CSA. By forming a complex of DBL3x with CSA oligosaccharides and determining its structure, we have identified the CSA binding site to be a cluster of conserved positively charged residues on subdomain 2 and subdomain 3. Mutation or chemical modification of lysine residues at the site markedly diminished CSA binding to DBL3x. The location of the CSA binding site is an important step forward in the molecular understanding of pregnancy-associated malaria and offers a new target for vaccine development.

The endothelial glycocalyx is well endowed with the glycosaminoglycans (GAGs) heparan sulfate, chondroitin sulfate and hyaluronan. The current studies aimed to assess the relative contributions of each of these GAGs to the thickness and permeability of the glycocalyx layer by direct enzymatic removal of each using micropipettes to infuse heparinase, chondroitinase and hyaluronidase into post-capillary venules of the intestinal mesentery of the rat. The relative losses of GAGs due to enzymatic removal were compared with stimulated shedding of glycans induced by superfusing the mesentery with 10(-)(7)M fMLP. Thickness of the glycocalyx was assessed by infiltration of the glycocalyx with circulating FITC labeled 70kDa dextran (Dx70) and measuring the distance from the dye front to the surface of the endothelium (EC), which averaged 463nm under control conditions. Reductions in thickness were 43.3%, 34.1% and 26.1% following heparinase, chondroitinase and hyaluronidase, respectively, and 89.7% with a mixture of all three enzymes. Diffusion coefficients of FITC in the glycocalyx were determined using a 1-D diffusion model. By comparison of measured transients in radial intensity of a bolus of FITC with that of a computational model a diffusion coefficient D was obtained. Values of D were obtained corresponding to the thickness of the layer demarcated by Dx70 (D(Dx70)), and a smaller sublayer 173nm above the EC surface (D(173)), prior to and following enzyme infusion and superfusion with fMLP. The magnitude of D(Dx70) was twice that of D(173) suggesting that the glycocalyx is more compact near the EC surface. Chondroitinase and hyaluronidase significantly increased both D(Dx70) and D(173). However, heparinase decreased D(Dx70), and did not induce any significant change for the D(173). These observations suggest that the three GAGs are not evenly distributed throughout the glycocalyx and that they each contribute to permeability of the glycocalyx to a differing extent. The fMLP-induced shedding caused a reduction in glycocalyx thickness (which may increase permeability) and as with heparinase, decreased the diffusion coefficient of solutes (which may decrease permeability). This behavior suggests that the removal of heparan sulfate may cause a collapse of the glycocalyx which counters decreases in thickness by compacting the layer to maintain a constant resistance to filtration.

Tenascin-R (TN-R), an extracellular matrix glycoprotein of the CNS, localizes to nodes of Ranvier and perineuronal nets and interacts in vitro with other extracellular matrix components and recognition molecules of the immunoglobulin superfamily. To characterize the functional roles of TN-R in vivo, we have generated mice deficient for TN-R by homologous recombination using embryonic stem cells. TN-R-deficient mice are viable and fertile. The anatomy of all major brain areas and the formation and structure of myelin appear normal. However, immunostaining for the chondroitin sulfate proteoglycan phosphacan, a high-affinity ligand for TN-R, is weak and diffuse in the mutant when compared with wild-type mice. Compound action potential recordings from optic nerves of mutant mice show a significant decrease in conduction velocity as compared with controls. However, at nodes of Ranvier there is no apparent change in expression and distribution of Na+ channels, which are thought to bind to TN-R via their beta2 subunit. The distribution of carbohydrate epitopes of perineuronal nets recognized by the lectin Wisteria floribunda or antibodies to the HNK-1 carbohydrate on somata and dendrites of cortical and hippocampal interneurons is abnormal. These observations indicate an essential role for TN-R in the formation of perineuronal nets and in normal conduction velocity of optic nerve.

In a search for small molecule antagonists of heparan sulfate, we examined the activity of bis-2-methyl-4-amino-quinolyl-6-carbamide, also known as surfen. Fluorescence-based titrations indicated that surfen bound to glycosaminoglycans, and the extent of binding increased according to charge density in the order heparin>> dermatan sulfate>> heparan sulfate>> chondroitin sulfate. All charged groups in heparin (N-sulfates, O-sulfates, and carboxyl groups) contributed to binding, consistent with the idea that surfen interacted electrostatically. Surfen neutralized the anticoagulant activity of both unfractionated and low molecular weight heparins and inhibited enzymatic sulfation and degradation reactions in vitro. Addition of surfen to cultured cells blocked FGF2-binding and signaling that depended on cell surface heparan sulfate and prevented both FGF2- and VEGF(165)-mediated sprouting of endothelial cells in Matrigel. Surfen also blocked heparan sulfate-mediated cell adhesion to the Hep-II domain of fibronectin and prevented infection by HSV-1 that depended on glycoprotein D interaction with heparan sulfate. These findings demonstrate the feasibility of identifying small molecule antagonists of heparan sulfate and raise the possibility of developing pharmacological agents to treat disorders that involve glycosaminoglycan-protein interactions.

A monoclonal antibody (HK-249) that recognizes a glucosamine sulfate alpha 1----4 glucuronic acid-containing determinant in heparan sulfate (HS) chains of a basement membrane-derived heparan sulfate proteoglycan identified and immunolocalized HS specifically to the amyloid deposits in neuritic plaques (NPs), congophilic angiopathy (CA), as well as in neurofibrillary tangles (NFTs) and non-tangle-bearing neurons in the brains of Alzheimer's and Down's syndrome (DS) patients. Ultrastructural immunohistochemistry demonstrated that HS within neurons of Alzheimer's disease (AD) brain was localized to lipofuscin granules, an aging pigment previously shown also to contain beta-amyloid protein (BAP). Heparan sulfate also was localized to neurite-containing, nonfibrillar 'primitive' plaques that also demonstrated positive BAP immunoreactivity in both AD and DS brains. Antibodies to laminin, fibronectin, and a chondroitin sulfate proteoglycan failed to show positive immunostaining of the HS-containing sites described above. Analysis of DS patients at different ages revealed that HS accumulated within neurons of the hippocampus and amygdala as early as 1 day after birth. Young age-matched controls did not demonstrate similar positive HS immunoreactivity in neurons, whereas positive immunostaining for HS was observed in other regions thought to normally contain HS. The earliest deposition of BAP was first observed as 'amorphous' or 'diffuse' cortical deposits in DS brain in patients aged 18 and 24 years before the accumulation of fibrillar amyloid (observed in DS patients who are 35 years and older). These cortical deposits also contained positive HS immunoreactivity, implying that HS accumulation in conjunction with the BAP is an early event that ultimately may contribute to the early age-related accumulation (ie, as early as 35 years of age in DS) of NPs, NFTs, and/or CA. Furthermore the colocalization of HS and BAP in a number of specific locales in AD and DS brain indicates a possible interaction between these two macromolecules that may be important in lesion development in these two diseases.

A series of disaccharides derived from chondroitin sulfate and heparin/heparan sulfate were derivatized at their reducing ends with a fluorophore 2-aminobenzamide to develop a sensitive microanalytical method for glycosaminoglycans. The resulting labeled compounds derived from chondroitin sulfate or heparin/heparan sulfate were well-separated and quantified by HPLC equipped with a fluorescence detector. The detection limit was a low picomole level. This method was applied to the analysis of the disaccharide composition of tetra- and hexasaccharides derived from chondroitin sulfate and heparin/heparan sulfate as well as these glycosaminoglycan polysaccharides. The method was also successfully applied to the exosequencing of chondrohexasaccharides, where the fluorophore-labeled oligosaccharides were degraded exolytically from the nonreducing ends using bacterial eliminases. The resultant labeled fragments were identified by HPLC.

Although the local environment is known to regulate neural stem cell (NSC) maintenance in the central nervous system, little is known about the molecular identity of the signals involved. Chondroitin sulfate proteoglycans (CSPGs) are enriched in the growth environment of NSCs both during development and in the adult NSC niche. In order to gather insight into potential biological roles of CSPGs for NSCs, the enzyme chondroitinase ABC (ChABC) was used to selectively degrade the CSPG glycosaminoglycans. When NSCs from mouse E13 telencephalon were cultivated as neurospheres, treatment with ChABC resulted in diminished cell proliferation and impaired neuronal differentiation, with a converse increase in astrocytes. The intrauterine injection of ChABC into the telencephalic ventricle at midneurogenesis caused a reduction in cell proliferation in the ventricular zone and a diminution of self-renewing radial glia, as revealed by the neurosphere-formation assay, and a reduction in neurogenesis. These observations suggest that CSPGs regulate neural stem/progenitor cell proliferation and intervene in fate decisions between the neuronal and glial lineage.

Proteoglycans that modulate the activities of growth factors, chemokines, and coagulation factors regulate in turn the vascular endothelium with respect to processes such as inflammation, hemostasis, and angiogenesis. Endothelial cell-specific molecule-1 is mainly expressed by endothelial cells and regulated by pro-inflammatory cytokines (Lassalle, P., Molet, S., Janin, A., Heyden, J. V., Tavernier, J., Fiers, W., Devos, R., and Tonnel, A. B. (1996) J. Biol. Chem. 271, 20458-20464). We demonstrate that this molecule is secreted as a soluble dermatan sulfate (DS) proteoglycan. This proteoglycan represents the major form either secreted by cell lines or circulating in the human bloodstream. Because this proteoglycan is specifically secreted by endothelial cells, we propose to name it endocan. The glycosaminoglycan component of endocan consists of a single DS chain covalently attached to serine 137. Endocan dose-dependently increased the hepatocyte growth factor/scatter factor (HGF/SF)-mediated proliferation of human embryonic kidney cells, whereas the nonglycanated form of endocan did not. Moreover, DS chains purified from endocan mimicked the endocan-mediated increase of cell proliferation in the presence of HGF/SF. Overall, our results demonstrate that endocan is a novel soluble dermatan sulfate proteoglycan produced by endothelial cells. Endocan regulates HGF/SF-mediated mitogenic activity and may support the function of HGF/SF not only in embryogenesis and tissue repair after injury but also in tumor progression.

Several sulfated polysaccharides (dextran sulfate, pentosan polysulfate, heparin) and copolymers of acrylic acid with vinylalcohol sulfate have proved to be potent inhibitors of human cytomegalovirus (CMV) infectivity in vitro. Sulfated alpha-cyclodextrins are only weak inhibitors of CMV. A close correlation was found between the 50% inhibitory concentrations of the sulfated polymers for CMV cytopathogenicity, virus-cell binding, and expression of immediate early antigens (IEA) in human embryonic lung (HEL) cells. CMV particles bound specifically to heparin-Sepharose. Sulfated polymers specifically eluted the virus particles from this matrix. Enzymatic digestion of cell surface heparan sulfate, but not of chondroitin sulfate, prevented the cells from being infected with CMV. Moreover, radiolabeled CMV bound efficiently to, and were infective for wild-type Chinese hamster ovary (CHO) cells, whereas virus binding to, and infection of, mutant CHO cell lines that were deficient in either all glycosaminoglycans or heparan sulfate only was significantly impaired. The mechanism of action of the sulfated polymers can be attributed to an inhibitory effect on the binding of CMV particles to the host cells. Presumably, the sulfated polymers interact with the viral envelope site(s) involved in the attachment of the CMV virions to cell surface heparan sulfate.

Expression cloning of cDNAs encoding a basic fibroblast growth factor (FGF) binding protein confirms previous hypotheses that this molecule is a cell-surface heparan sulfate proteoglycan. A cDNA library constructed from a hamster kidney cell line rich in FGF receptor activity was transfected into a human lymphoblastoid cell line. Clones expressing functional basic FGF binding proteins at their surfaces were enriched by panning on plastic dishes coated with human basic FGF. The amino acid sequence deduced from the isolated cDNAs revealed several interesting features, including hydrophobic signal and transmembrane domains that flank an extracellular region containing six potential attachment sites for glycosaminoglycan side chains. The structure also contains a short hydrophilic cytoplasmic tail sequence homologous to previously reported actin binding domains. Binding of basic FGF to cells expressing the binding protein could be inhibited by heparin and heparan sulfate but not by chondroitin sulfate, dermatan sulfate, or keratan sulfate. In addition to binding basic FGF, this protein or related surface proteins may function as an initial cellular attachment site for other growth factors and for viruses, such as herpes simplex virus.

Myo-intimal proteoglycan metabolism is thought to be important in blood vessel homeostasis, blood clotting, atherogenesis, and atherosclerosis. Human platelet-derived transforming growth factor type beta (TGF-beta) specifically stimulated synthesis of at least two types of chondroitin sulfate proteoglycans in nonproliferating human adult arterial smooth muscle cells in culture. Stimulation of smooth muscle cell proteoglycan synthesis by smooth muscle cell growth promoters (epidermal growth factor, platelet-derived growth factor, and heparin-binding growth factors) was less than 20% of that elicited by TGF-beta. TGF-beta neither significantly stimulated proliferation of quiescent smooth muscle cells nor inhibited proliferating cells. The extent of TGF-beta stimulation of smooth muscle cell proteoglycan synthesis was similar in both nonproliferating and growth-stimulated cells. TGF-beta, which is a reversible inhibitor of endothelial cell proliferation, had no comparable effect on endothelial cell proteoglycan synthesis. These results are consistent with the hypothesis that TGF-beta is a cell-type-specific regulator of proteoglycan synthesis in human blood vessels and may contribute to the myo-intimal accumulation of proteoglycan in atherosclerotic lesions.

Selectin-mediated binding of tumor cells to platelets, leukocytes, and vascular endothelium may regulate their hematogenous spread in the microvasculature. We recently reported that CD44 variant isoforms (CD44v) on LS174T colon carcinoma cells possess selectin binding activity. Here we extended those findings by showing that T84 and Colo205 colon carcinoma cells bind selectins via sialidase-sensitive O-linked glycans presented on CD44v, independent of heparan and chondroitin sulfate. To assess the functional role of CD44v in selectin-mediated binding, we quantified the adhesion to selectins of T84 cell subpopulations sorted based on their CD44 expression levels and stable LS174T cell lines generated using CD44 short hairpin RNA. High versus low CD44-expressing T84 cells tethered more efficiently to P- and L-selectin, but not E-selectin, and rolled more slowly on P- and E-selectin. Knocking down CD44 expression on LS174T cells inhibited binding to P-selectin and increased rolling velocities over P- and L-selectin relative to control-transfected cells, without affecting tethering and rolling on E-selectin, however. Blot rolling analysis revealed the presence of alternative sialylated glycoproteins with molecular masses of approximately 170 and approximately 130 kDa, which can mediate selectin binding in CD44-knockdown cells. Heparin diminishes the avidity of colon carcinoma cells for P- and L-selectin, which may compromise integrin-mediated firm adhesion to host cells and mitigate metastasis. Our finding that CD44v is a functional P-selectin ligand on colon carcinoma provides a novel perspective on the enhanced metastatic potential associated with tumor CD44v overexpression and the role of selectins in metastasis.

Astrocytes, oligodendrocytes, and oligodendrocyte/type 2 astrocyte progenitors (O2A cells) can all produce molecules that inhibit axon regeneration. We have shown previously that inhibition of axon growth by astrocytes involves proteoglycans. To identify inhibitory mechanisms, we created astrocyte cell lines that are permissive or nonpermissive and showed that nonpermissive cells produce inhibitory chondroitin sulfate proteoglycans (CS-PGs). We have now tested these cell lines for the production and inhibitory function of known large CS-PGs. The most inhibitory line, Neu7, produces three CS-PGs in much greater amounts than the other cell lines: NG2, versican, and the CS-56 antigen. The contribution of NG2 to inhibition by the cells was tested using a function-blocking antibody. This allowed increased growth of dorsal root ganglion (DRG) axons over Neu7 cells and matrix and greatly increased the proportion of cortical axons able to cross from permissive A7 cells onto inhibitory Neu7 cells; CS-56 antibody had a similar effect. Inhibitory fractions of conditioned medium contained NG2 coupled to CS glycosaminoglycan chains, whereas noninhibitory fractions contained NG2 without CS chains. Enzyme preparations that facilitated axon growth in Neu7 cultures were shown to either degrade the NG2 core protein or remove CS chains. Versican is present as patches on Neu7 monolayers, but DRG axons do not avoid these patches. Therefore, NG2 appears to be the major axon-inhibitory factor made by Neu7 astrocytes. In the CNS, NG2 is expressed by O2A cells, which react rapidly after injury to produce a dense NG2-rich network, and by some reactive astrocytes. Our results suggest that NG2 may be a major obstacle to axon regeneration.

Pleiotrophin/heparin-binding growth-associated molecule (HB-GAM) is a specific ligand of protein tyrosine phosphatase zeta (PTPzeta)/receptor-like protein tyrosine phosphatase beta (RPTPbeta) expressed in the brain as a chondroitin sulfate proteoglycan. Pleiotrophin and PTPzeta isoforms are localized along the radial glial fibers, a scaffold for neuronal migration, suggesting that these molecules are involved in migratory processes of neurons during brain development. In this study, we examined the roles of pleiotrophin-PTPzeta interaction in the neuronal migration using cell migration assay systems with glass fibers and Boyden chambers. Pleiotrophin and poly-L-lysine coated on the substratums stimulated cell migration of cortical neurons, while laminin, fibronectin, and tenascin exerted almost no effect. Pleiotrophin-induced and poly-L-lysine-induced neuronal migrations showed significant differences in sensitivity to various molecules and reagents. Polyclonal antibodies against the extracellular domain of PTPzeta, PTPzeta-S, an extracellular secreted form of PTPzeta, and sodium vanadate, a protein tyrosine phosphatase inhibitor, added into the culture medium strongly suppressed specifically the pleiotrophin-induced neuronal migration. Furthermore, chondroitin sulfate C but not chondroitin sulfate A inhibited pleiotrophin-induced neuronal migration, in good accordance with our previous findings that chondroitin sulfate constitutes a part of the pleiotrophin-binding site of PTPzeta, and PTPzeta-pleiotrophin binding is inhibited by chondroitin sulfate C but not by chondroitin sulfate A. Immunocytochemical analysis indicated that the transmembrane forms of PTPzeta are expressed on the migrating neurons especially at the lamellipodia along the leading processes. These results suggest that PTPzeta is involved in the neuronal migration as a neuronal receptor of pleiotrophin distributed along radial glial fibers.

Bone resorption in balance with bone formation is vital for the maintenance of the skeleton and is mediated by osteoclasts. Cathepsin K is the predominant protease in osteoclasts that degrades the bulk of the major bone forming organic component, type I collagen. Although the potent collagenase activity of cathepsin K is well known, its mechanism of action remains elusive. Here, we report a cathepsin K-specific complex with chondroitin sulfate, which is essential for the collagenolytic activity of the enzyme. The complex is an oligomer consisting of five cathepsin K and five chondroitin sulfate molecules. Only the complex exhibits potent triple helical collagen-degrading activity, whereas monomeric cathepsin K has no collagenase activity. The primary substrate specificity of cathepsin K is not altered by complex formation, suggesting that the protease-chondroitin sulfate complex primarily facilitates the destabilization and/or the specific binding of the triple helical collagen structure. Inhibition of complex formation leads to the loss of collagenolytic activity but does not impair the proteolytic activity of cathepsin K toward noncollagenous substrates. The physiological relevance of cathepsin K complexes is supported by the findings that (i) the content of chondroitin sulfate present in bone and accessible to cathepsin K activity is sufficient for complex formation and (ii) Y212C, a cathepsin K mutant that causes pycnodysostosis (a bone sclerosing disorder) and that has no collagenase activity but remains potent as a gelatinase, is unable to form complexes. These findings reveal a novel mechanism of bone collagen degradation and suggest that targeting cathepsin K complex formation would be an effective and specific treatment for diseases with excessive bone resorption such as osteoporosis.

A comparative analysis was carried out of heparan sulfate (HS) and chondroitin sulfate (CS) chains of the ectodomains of hybrid type transmembrane proteoglycans, syndecan-1 and -4, synthesized simultaneously by normal murine mammary gland epithelial cells. Although the HS chains were structurally indistinguishable, intriguingly the CS chains were structurally and functionally distinct, probably reflecting the differential regulation of sulfotransferases involved in the synthesis of HS and CS. The CS chains of the two syndecans comprised nonsulfated, 4-O-, 6-O-, and 4,6-O-disulfated N-acetylgalactosamine-containing disaccharide units and were significantly different, with a higher degree of sulfation for syndecan-4. Functional analysis using a BIAcore system showed that basic fibroblast growth factor (bFGF) specifically bound only to the HS chains of both syndecans, whereas midkine (MK) and pleiotrophin (PTN) bound not only to the HS but also to the CS chains. Stronger binding of MK and PTN to the CS chains of syndecan-4 than those of syndecan-1 was revealed, supporting the structural and functional differences. Intriguingly, removal of the CS chains decreased the association and dissociation rate constants of MK, PTN, and bFGF for both syndecans, suggesting the simultaneous binding of these growth factors to both types of chains, producing a ternary complex that transfers the growth factors to the corresponding cell surface receptors more efficiently compared with the HS chains alone. The involvement of the core protein was also shown in the binding of MK and PTN to syndecan-1, suggesting the possibility of cooperation with the HS and/or CS chains in the binding of these growth factors and their delivery to the cell surface receptors.

Biglycan is a Class I Small Leucine Rich Proteoglycans (SLRP) that is localized on human chromosome Xq28-ter. The conserved nature of its intron-exon structure and protein coding sequence compared to decorin (another Class I SLRP) indicates the two genes may have arisen from gene duplication. Biglycan contains two chondroitin sulfate glycosaminoglycan (GAG) chains attached near its NH(2) terminus making it different from decorin that has only one GAG chain. To determine the functions of biglycan in vivo, transgenic mice were developed that were deficient in the production of the protein (knockout). These mice acquire diminished bone mass progressively with age. Double tetracycline-calcein labeling revealed that the biglycan deficient mice are defective in their capacity to form bone. Based on this observation, we tested the hypothesis that the osteoporosis-like phenotype is due to defects in cells critical to the process of bone formation. Our data shows that biglycan deficient mice have diminished capacity to produce marrow stromal cells, the bone cell precursors, and that this deficiency increases with age. The cells also have reduced response to tranforming growth factor-beta (TGF-beta), reduced collagen synthesis and relatively more apoptosis than cells from normal littermates. In addition, calvaria cells isolated from biglycan deficient mice have reduced expression of late differentiation markers such as bone sialoprotein and osteocalcin and diminished ability to accumulate calcium judged by alizerin red staining. We propose that any one of these defects in osteogenic cells alone, or in combination, could contribute to the osteoporosis observed in the biglycan knockout mice. Other data suggests there is a functional relationship between biglycan and bone morphogenic protein-2/4 (BMP 2/4) action in controlling skeletal cell differentiation. In order to test the hypothesis that functional compensation can occur between SLRPs, we created mice deficient in biglycan and decorin. Decorin deficient mice have normal bone mass while the double biglycan/decorin knockout mice have more severe osteopenia than the single biglycan indicating redundancy in SLRP function in bone tissue. To further determine whether compensation could occur between different classes of SLRPs, mice were generated that are deficient in both biglycan (class I) and fibromodulin, a class II SLRP highly expressed in mineralizing tissue. These doubly deficient mice had an impaired gait, ectopic calcification of tendons and premature osteoarthritis. Transmission electron microscopy analysis showed that like the decorin and biglycan knockouts, they have severely disturbed collagen fibril structures. Biomechanical analysis of the affected tendons showed they were weaker compared to control animals leading to the conclusion that instability of the joints could be the primary cause of all the skeletal defects observed in the fibromodulin/biglycan knockout mice. These studies present important new animal models for musculoskeletal diseases and provide the opportunity to characterize the network of signals that control tissue integrity and function through SLRP activity.

Coating of orthopaedic implants with extracellular bone matrix components was performed to enhance bone healing. Titanium pins of 0.8mm diameter were coated with type I collagen (Ti/Coll), RGD peptide (Ti/RGD) or type I collagen and chondroitin sulfate (Ti/Coll/CS). Uncoated pins (Ti) served as control. The pins were inserted as intramedullary nails into the tibia of male adult Wistar rats. Six specimens of each group were retrieved at 4, 7, 14 and 28 days. All implants healed uneventfully without adverse reactions. ED 1-positive macrophages appeared in higher numbers around Ti/RGD at day 4 and around Ti at day 14 after implantation (p < 0.05). TRAP-positive osteoclasts and precursors were abundant around Ti/Coll/CS at day 7 (p < 0.05). A significant increase in osteopontin-positive osteoblasts was seen around Ti/Coll/CS implants at days 7 and 14, and around Ti/RGD at day 14 (p < 0.05). At day 28, 62% of Ti, 76% of Ti/Coll, 85%* of Ti/RGD and 89%* of Ti/CoIl/CS (*p < 0.05) implants were covered with newly formed lamellar bone. The addition of extracellular matrix components significantly enhances bone remodelling in the early stages of bone healing around Ti implants, eventually leading to increased new bone formation at the implant surface after 4 weeks.

Extracellular matrix molecules accumulate around central nervous system neurons during postnatal development, forming so-called perineuronal nets (PNNs). PNNs play a role in restricting plasticity at the end of critical periods. In the adult rat cerebellum, PNNs are found around large, deep cerebellar nuclei (DCN) neurons and Golgi neurons and are composed of chondroitin sulfate proteoglycans (CSPGs), tenascin-R (TN-R), hyaluronan (HA), and link proteins, such as cartilage link protein 1 (Crtll). Granule cells and Purkinje cells are surrounded by a partially organized matrix. Both glial cells and neurons surrounded by PNNs are the site of synthesis of some CSPGs and of TN-R, but only neurons produce HA synthetic enzymes (HASs), thus HA, and link proteins, which are scaffolding molecules for an organized matrix. To elucidate the mechanisms of formation of PNNs, we analyzed by immunohistochemistry and in situ hybridization which PNN components are upregulated during PNN formation in rat cerebellar postnatal development and what cell types express them. We observed that Wisteria floribunda agglutinin-binding PNNs develop around DCN neurons from postnatal day (P)7 and around Golgi neurons from P14. At the same time as their PNNs start to form, these neurons upregulate aggrecan, Crtll, and HASs mRNAs. However, Crtll is the only PNN component to be expressed exclusively in neurons surrounded by PNNs. The other link protein that shows a perineuronal net pattern in the DCN, Bral2, is upregulated later during development. These data suggest that aggrecan, HA, and, particularly, Crtll might be crucial elements for the initial assembly of PNNs.

We report the identification and DNA sequence of a chondroitin sulfate proteoglycan core protein cDNA. A cDNA clone, pPG1, was selected from a rat yolk sac tumor poly(A)+RNA-derived cDNA library by using synthetic oligonucleotides predicted from the NH2-terminal peptide sequence of the mature chondroitin sulfate proteoglycan. The resulting sequence analysis demonstrated that the 874-base-pair pPG1 clone contained the complete coding region of the mature proteoglycan core protein as well as 5' and 3' flanking sequences. The 104 amino acid proteoglycan core protein sequence reveals that the core protein is composed of three regions, the most striking of which is the central 49 amino acid region composed of alternating serine and glycine residues. This region clearly functions as the acceptor site for the attachment of chondroitin sulfate side chains. The serine-glycine repeat region is flanked by a 14 amino acid NH2-terminal region identical to the NH2-terminal sequence of the proteoglycan obtained by amino acid sequencing and a 41 amino acid COOH-terminal region. RNA transfer blot hybridizations of poly(A)+ mRNA from rat yolk sac tumor cells with nick-translated pPG1 reveal a single mRNA of approximately equal to 1300 nucleotides. The possibility of detecting mRNAs and genomic sequences for other proteoglycans with a serine-glycine repeat by using this cDNA clone is discussed.