In pregnant women infected with Plasmodium falciparum, the infected red blood cells (IRBCs) selectively accumulate in the intervillous spaces of placenta, leading to poor fetal outcome and severe health complications in the mother. Although chondroitin 4-sulfate is known to mediate IRBC adherence to placenta, the natural receptor has not been identified. In the present study, the chondroitin sulfate proteoglycans (CSPGs) of human placenta were purified and structurally characterized, and adherence of IRBCs to these CSPGs investigated. The data indicate that the placenta contains three distinct types of CSPGs: significant quantities of uniquely low sulfated, extracellular CSPGs localized in the intervillous spaces, minor amounts of two cell-associated CSPGs, and major amounts of dermatan sulfate-like CSPGs of the fibrous tissue. Of the various CSPGs isolated from the placenta, the low sulfated CSPGs of the intervillous spaces most efficiently bind IRBCs. Based on IRBC adherence capacities and localization patterns of various CSPGs, we conclude that the CSPGs of the intervillous spaces are the receptors for placental IRBC adherence. The identification and characterization of these CSPGs provide a valuable tool for understanding the precise molecular interactions involved in placental IRBC adherence and for the development of therapeutic strategies for maternal malaria. In the accompanying paper (Alkhalil, A., Achur, R. N., Valiyaveettil, M., Ockenhouse, C. F., and Gowda, D. C. (2000) J. Biol. Chem. 275, 40357-40364), we report the structural requirements for the IRBC adherence.

We studied the localization of latent transforming growth factor-beta 1 (TGF-beta 1) and its binding protein (LTBP-1) in the extracellular matrix of cultured human fibroblasts by immunofluorescence and immunoelectron microscopy. Immunofluorescence of confluent fibroblast cultures indicated that LTBP-1 localizes to extracellular fibrillar structures resembling fibronectin-collagen matrix. Similar fibrillar structures were detected in cells stained with antibodies specific for TGF-beta 1 propeptide (beta 1-LAP). Both LTBP-1 and beta 1-LAP colocalized with fibronectin in double immunofluorescence analysis. These fibrillar structures were resistant to extraction with sodium deoxycholate, which is further evidence that LTBP-1 and large latent TGF-beta 1 complexes are integral components of the extracellular matrix. SV-40-transformed human fibroblasts lacked extracellular LTBP-1 fibers. EM analysis revealed approximately 10-nm-thick microfibrils that were labeled by anti-LTBP at 90-140-nm intervals. In addition, LTBP-1 was found in structures that were heavily labeled for fibronectin. The accumulation of LTBP-1 in the fibronectin matrix could be reconstituted in vitro. When isolated matrix components were immobilized on nitrocellulose and incubated with fibroblast conditioned medium, LTBP-1 from the medium associated with cellular fibronectin but not with heparan or chondroitin sulfate, vitronectin, tenascin, laminin, or collagen I or IV. The association of LTBP-1 with cellular fibronectin was abolished by treatment of the medium with plasmin, which cleaves LTBP-1 and inhibits its assembly to matrix. The present results indicate that latent TGF-beta 1 complexes are components of the extracellular matrix and suggest that alterations of the pericellular matrix could result in aberrant TGF-beta signaling.

The contribution of chondroitin sulfate proteoglycan (CSPG) in the suppression of axonal growth in rat spinal cord has been examined by means of an in vitro bioassay in which regenerating neurons are grown on tissue section substrata. Dissociated embryonic chick dorsal root ganglionic neurons were grown on normal and injured adult spinal cord tissue sections treated with chondroitinases. Neuritic growth on normal spinal cord tissue was meager. However, both the percentage of neurons with neurites and the average neurite length were substantially greater on sections treated with chondroitinase ABC. Enzymes that specifically degraded dermatan sulfate or hyaluronan were ineffective. Neuritic growth was significantly greater on injured (compared to normal) spinal cord and a further dramatic increase resulted from chondroitinase ABC treatment. Neurites grew equally within white and gray matter regions after chondroitinase treatment. Observed increases in neurite outgrowth on chondroitinase-treated tissues were largely inhibited in the presence of function-blocking laminin antibodies. These findings indicate that inhibitory CSPG is widely distributed and predominant in both normal and injured spinal cord tissues. Additionally, inhibitory CSPG is implicated in negating the potential stimulatory effects of laminin that might otherwise support spinal cord regeneration.

C-terminal truncation of ADAMTS-4 from the p68 form to the p53 form is required for activation of its capacity to cleave the Glu(373)-Ala(374) interglobular domain bond of aggrecan. In transfected human chondrosarcoma cells, this process is not autoproteolytic because the same products form with an inactive mutant of ADAMTS4 (a disintegrin and metalloproteinase with thrombospondin-like motif 4) and truncation is completely blocked by tissue inhibitor of metalloproteinase-1. Instead, activation can be mediated by glycosylphosphatidyl inositol-anchored membrane type 4-matrix metalloproteinase (MT4-MMP, MMP-17) because co-transfection with the active form of MT4-MMP markedly enhanced activation, whereas an inactive mutant of MT4-MMP was ineffective. Treatment of co-transfected cells with phosphatidylinositol-specific phospholipase C liberated the complex of MT4-MMP and p68 ADAMTS4 from the cell membrane, but the p53 ADAMTS4 remained associated. Specific glycosaminoglycan lyase digestions, followed by product analyses using fluorescence-assisted carbohydrate electrophoresis and immunoprecipitation experiments, showed that the p53 form is associated with syndecan-1 through both chondroitin sulfate and heparan sulfate. We conclude that ADAMTS-4 activation in this cell system involves the coordinated activity of both glycosylphosphatidyl inositol-anchored MT4-MMP and the proteoglycan form of syndecan-1 on the cell surface.

A line of null mice has been produced which fails to express the transmembrane chondroitin sulfate proteoglycan NG2. Homozygous NG2 null mice do not exhibit gross phenotypic differences from wild-type mice, suggesting that detailed analyses are required to detect subtle alterations caused by the absence of NG2. Accordingly, dissociated cultures of aortic smooth muscle cells from null mice were compared to parallel cultures from wild-type mice for their ability to proliferate and migrate in response to specific growth factors. Both null and wild-type smooth muscle cells exhibited identical abilities to proliferate and migrate in response to PDGF-BB. In contrast, only the wild-type cells responded to PDGF-AA in both types of assays. NG2 null cells failed to proliferate or migrate in response to PDGF-AA, implying a defect in the signaling cascade normally initiated by activation of the PDGF (alpha)-receptor. In agreement with this idea, activation of the extracellular signal-regulated kinase (ERK) in response to PDGF-AA treatment occured only in wild-type cells. Failure to observe autophosphorylation of the PDGF (alpha)-receptor in PDGF-AA-treated null cells indicates that the absence of NG2 causes a defect in signal transduction at the level of (alpha)-receptor activation.

BACKGROUND

Articular cartilage is a highly functional tissue which covers the ends of long bones and serves to ensure proper joint movement. A tissue engineering approach that recapitulates the developmental characteristics of articular cartilage can be used to examine the maturation and degeneration of cartilage and produce fully functional neotissue replacements for diseased tissue.

RESULTS

This study examined the development of articular cartilage neotissue within a self-assembling process in two phases. In the first phase, articular cartilage constructs were examined at 1, 4, 7, 10, 14, 28, 42, and 56 days immunohistochemically, histologically, and through biochemical analysis for total collagen and glycosaminoglycan (GAG) content. Based on statistical changes in GAG and collagen levels, four time points from the first phase (7, 14, 28, and 56 days) were chosen to carry into the second phase, where the constructs were studied in terms of their mechanical characteristics, relative amounts of collagen types II and VI, and specific GAG types (chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate, and hyaluronan). Collagen type VI was present in initial abundance and then localized to a pericellular distribution at 4 wks. N-cadherin activity also spiked at early stages of neotissue development, suggesting that self-assembly is mediated through a minimization of free energy. The percentage of collagen type II to total collagen significantly increased over time, while the proportion of collagen type VI to total collagen decreased between 1 and 2 wks. The chondroitin 6- to 4- sulfate ratio decreased steadily during construct maturation. In addition, the compressive properties reached a plateau and tensile characteristics peaked at 4 wks.

CONCLUSIONS

The indices of cartilage formation examined in this study suggest that tissue maturation in self-assembled articular cartilage mirrors known developmental processes for native tissue. In terms of tissue engineering, it is suggested that exogenous stimulation may be necessary after 4 wks to further augment the functionality of developing constructs.

Serum concentrations of specific cartilage and bone molecules reflecting tissue turnover were measured in two well-defined patient groups with early rheumatoid arthritis with distinctly different disease outcome to see if early differences in their levels are prognostic of the rate of joint destruction. Compared with a matched normal population, increased concentrations of cartilage oligomeric matrix protein (COMP) were found in all patients who developed rapid hip joint destruction. In contrast, levels of a putative marker of cartilage aggrecan synthesis, the chondroitin sulfate epitope 846, were increased only in patients with slow joint destruction. Levels of bone sialoprotein (BSP) were increased in both groups, as were levels of the C-propeptide of type II procollagen (CPII), a marker of collagen II synthesis. The increased concentrations of the 846 epitope in patients with slow joint destruction suggest increased aggrecan synthesis. The low levels of the 846 epitope in patients with rapid joint destruction, concomitant with elevated levels of CPII, suggest a selective increase in collagen synthesis. The elevated BSP levels indicate an increased bone turnover in both groups. Thus elevated serum levels of COMP may indicate an unfavorable prognosis for rapid joint destruction, whereas elevated 846 epitope indicates a more favorable prognosis.

The embryonic corneal epithelium synthesizes both collagen and chondroitin sulfate and excretes them across the basement membrane into the subepithelial space where they assemble into a spiraling orthogonal matrix of fibrils. The assembly of collagen into fibrils is first apparent at the outer face of the basement membrane in a region of ordered chondroitin sulfate molecules. Hyaluronate, another morphogenetically important corneal macromolecule, is produced at these early stages only by the inner endothelium. These correlated biosynthetic and ultrastructural data demonstrate discrete macromolecular products of the two corneal epithelia with differing morphogenetic properties and functions.

Grafting of Schwann cell-seeded channels into hemisected adult rat thoracic spinal cords has been tested as a strategy to bridge the injured cord. Despite success in guiding axonal growth into the graft, regeneration across the distal graft-host interface into the host spinal cord was limited. We hypothesized that chondroitin sulfate (CS) glycoforms deposited at the gliotic front of the interface constitute a molecular barrier to axonal growth into the host cord. Because CS glycoforms deposited by purified astrocytes in vitro were removable by digestion with chondroitinase ABC, we attempted to achieve likewise by infusion of the enzyme to the host side of the interface. By 1 month post-treatment, significant numbers of regenerating axons crossed an interface that was subdued in macrophage/microglia reaction and decreased in CS-immunopositivity. The axons extended as far into the caudal cord as 5 mm, in contrast to nil in vehicle-infused controls. Fascicular organizations of axon-Schwann cell units within the regenerated tissue cable were better-preserved in enzyme-treated cords than in vehicle-infused controls. We conclude that CS glycoforms deposited during gliosis at the distal graft-host interface could be cleared by the in vivo action of chondroitinase ABC to improve prospects of axonal regeneration into the host spinal cord.

Half of patients with muscle-invasive bladder cancer develop metastatic disease, and this is responsible for most of the deaths from this cancer. Low expression of RhoGTP dissociation inhibitor 2 (RhoGDI2; also known as ARHGDIB and Ly-GDI) is associated with metastatic disease in patients with muscle-invasive bladder cancer. Moreover, a reduction in metastasis is observed upon reexpression of RhoGDI2 in xenograft models of metastatic cancer. Here, we show that RhoGDI2 suppresses lung metastasis in mouse models by reducing the expression of isoforms V1 and V3 of the proteoglycan versican (VCAN; also known as chondroitin sulfate proteoglycan 2 [CSPG2]). In addition, we found that high versican levels portended poor prognosis in patients with bladder cancer. The functional importance of tumor expression of versican in promoting metastasis was established in in vitro and in vivo studies in mice that implicated a role for the chemokine CCL2 (also known as MCP1) and macrophages. Further analysis indicated that RhoGDI2 suppressed metastasis by altering inflammation in the tumor microenvironment. In summary, we demonstrate what we believe to be a new mechanism of metastasis suppression that works by reducing host responses that promote metastatic colonization of the lung. Therapeutic targeting of these interactions may provide a novel adjuvant strategy for delaying the appearance of clinical metastasis in patients.

The early events in neoplastic transformation can be understood only by comparison of the neoplastic cell with its nontransformed counterpart. The most common central nervous system gliomas traditionally are thought to arise from mature astrocytes and oligodendrocytes. We examined the possibility that gliomas arise from a population of glia that has properties of oligodendrocyte progenitors. These glial cells express the NG2 chondroitin sulfate proteoglycan and the alpha receptor of platelet-derived growth factor in vivo. We identified NG2 and the alpha receptor of platelet-derived growth factor expression in tissue from seven of seven oligodendrogliomas, three of three pilocytic astrocytomas, and one of five glioblastoma multiforme. These data provide evidence that glial tumors arise from glial progenitor cells. Molecules expressed by these progenitor cells should be considered as targets for novel therapeutics.

Glycosaminoglycans (GAGs) play a critical role in binding and activation of growth factors involved in cell signaling critical for developmental biology. The biosynthetic pathways for GAGs have been elucidated over the past decade and now analytical methodology makes it possible to determine GAG composition in as few as 10 million cells. A glycomics approach was used to examine GAG content, composition, and the level of transcripts encoding for GAG biosynthetic enzymes as murine embryonic stem cells (mESCs) differentiate to embryoid bodies (EBs) and to extraembryonic endodermal cells (ExE) to better understand the role of GAGs in stem cell differentiation. Hyaluronan synthesis was enhanced by 13- and 24-fold, most likely due to increased expression of hyaluronan synthase-2. Chondroitin sulfate (CS)/dermatan sulfate (DS) synthesis was enhanced by 4- and 6-fold, and heparan sulfate (HS) synthesis was enhanced by 5- and 8-fold following the transition from mESC to EB and ExE. Transcripts associated with the synthesis of the early precursors were largely unaltered, suggesting other factors account for enhanced GAG synthesis. The composition of both CS/DS and HS also changed upon differentiation. Interestingly, CS type E and highly sulfated HS both increase as mESCs differentiate to EBs and ExE. Differentiation was also accompanied by enhanced 2-sulfation in both CS/DS and HS families. Transcript levels for core proteins generally showed increases or remained constant upon mESC differentiation. Finally, transcripts encoding selected enzymes and isoforms, including GlcNAc-4,6-O-sulfotransferase, C5-epimerases, and 3-O-sulfotransferases involved in late GAG biosynthesis, were also enriched. These biosynthetic enzymes are particularly important in introducing GAG fine structure, essential for intercellular communication, cell adhesion, and outside-in signaling. Knowing the changes in GAG fine structure should improve our understanding the biological properties of differentiated stem cells.

The cell line HMC-1, derived from a patient with mast cell leukaemia, is the only established cell line exhibiting a phenotype similar to that of human mast cells. This paper reports on a detailed characterization of the expression of a panel of markers for various types of immature and mature haematopoietic cells in the HMC-1. We also studied the potential of HMC-1 to differentiate upon treatment with conditioned media from the human T-cell line Mo, retinoic acid or DMSO. HMC-1 was found to express several mast cell-related markers. A high expression of Kit, the receptor for stem-cell factor, was detected. The majority of the cells were stained with a MoAb against the mast cell-specific serine protease tryptase. Of particular interest was the finding that beta-tryptase mRNA, but not alpha-tryptase mRNA, was expressed in HMC-1. Using enzyme-histochemistry we were able to show that the beta-tryptase was enzymatically active, indicating that tryptase can form active homotetramers. Both heparin and chondroitin sulfate were found to be present in approximately equal amounts. HMC-1 lacked surface expression of the high-affinity IgE receptor, which was confirmed by the absence of mRNA of the alpha- and beta-chains of the IgE-receptor complex. However, a strong expression of the gamma-chain of the IgE-receptor complex was detected. A positive staining of the monocyte/macrophage marker CD68 was obtained, as well as a strong hybridization signal for the eosinophilic/basophilic-related differentiation marker the Charcot-Leyden crystal. Treatment of HMC-1 with conditioned media from the human T-cell line Mo, retinoic acid or DMSO induced only moderate changes in the surface or intracellular expression of the studied markers. The agents tested neither induced any of the monocyte/granulocyte markers examined, nor expression of the Fc epsilon RI alpha-chain.

We have previously identified megakaryocyte stimulating factor (MSF) gene expression by synovial fibroblasts as the origin of lubricin in the synovial cavity. Lubricin is a mucinous glycoprotein responsible for the boundary lubrication of articular cartilage. MSF has a significant homology to vitronectin and is composed of 12 exons. RNA was purified from human synovial fibroblasts and articular chondrocytes grown in vitro from tissue explants obtained from subjects without degenerative joint disease. RT-PCR was used with multiple complimentary primer pairs spanning the central mucin expressing exon 6 of the MSF gene and individual exons on both the N- and C-terminal sides of exon 6. Exons 2, 4 and 5 appear to be variably expressed by synovial fibroblasts and articular chondrocytes. Lubricating mucin, in the form of MSF, is expressed by both chondrocytes and synovial fibroblasts in vitro. Both lubricin and superficial zone protein (SZP), a related proteoglycan, share a similar primary structure but could differ in post-translational modifications with O-linked oligosaccharides which are predominant in lubricin and with limited amounts chondroitin and keratan sulfate found in SZP. Since most of the MSF exons are involved in the expression of lubricating mucin, a strong homology to vitronectin persists. It is therefore appropriate to consider that both SZP and lubricin occupy a new class of biomolecules termed tribonectins. Screening of a human genome bacterial artificial chromsome (BAC) library with a cDNA primer pair complimentary for exon 6 identified two clones. Both clones were complimentary for chromosome 1q25 by in situ hybridization. This same locus was previously implicated in camptodactyl-arthropathy-pericarditis syndrome (CAP) by genetic mapping. It is hypothesized that CAP, a large joint arthropathy, may be associated with ineffective boundary lubrication provided by synovial fluid.

Highly sulfated proteoglycans are correlated with axon boundaries in the developing central nervous system which suggests that these molecules affect neural pattern formation. In the developing mammalian retina, gradual regression of chondroitin sulfate may help control the onset of ganglion cell differentiation and initial direction of their axons. Changes induced by the removal of chondroitin sulfate from intact retinas in culture confirm the function of chondroitin sulfate in retinal histogenesis.

Endothelial cells (ECs) are covered by a surface glycocalyx layer that forms part of the barrier and mechanosensing functions of the blood-tissue interface. Removal of albumin in bathing media induces collapse or shedding of the glycocalyx. The electrostatic interaction between arginine residues on albumin, and negatively charged glycosaminoglycans (GAGs) in the glycocalyx have been hypothesized to stabilize the glycocalyx structure. Because albumin is one of the primary carriers of the phospholipid sphingosine-1-phosphate (S1P), we evaluated the alternate hypothesis that S1P, acting via S1P1 receptors, plays the primary role in stabilizing the endothelial glycocalyx. Using confocal microscopy on rat fat-pad ECs, we demonstrated that heparan sulfate (HS), chondroitin sulfate (CS), and ectodomain of syndecan-1 were shed from the endothelial cell surface after removal of plasma protein but were retained in the presence of S1P at concentrations of >100 nM. S1P1 receptor antagonism abolished the protection of the glycocalyx by S1P and plasma proteins. S1P reduced GAGs released after removal of plasma protein. The mechanism of protection from loss of glycocalyx components by S1P-dependent pathways was shown to be suppression of metalloproteinase (MMP) activity. General inhibition of MMPs protected against loss of CS and syndecan-1. Specific inhibition of MMP-9 and MMP-13 protected against CS loss. We conclude that S1P plays a critical role in protecting the glycocalyx via S1P1 and inhibits the protease activity-dependent shedding of CS, HS, and the syndecan-1 ectodomain. Our results provide new insight into the role for S1P in protecting the glycocalyx and maintaining vascular homeostasis.

In recent years, the endothelial cell surface glycocalyx has emerged as a structure of fundamental importance to a broad range of phenomena that determine cardiovascular health and disease. This new understanding of the functional significance of the glycocalyx has been made possible through recently developed experimental techniques using intravital microscopy that are capable of directly probing the glycocalyx in vivo. Using fluorescent microparticle image velocimetry in venules and endothelialized cylindrical collagen microchannels, we show that the hydrodynamically relevant endothelial cell glycocalyx surface layer observed in microvessels in vivo (0.52+/-0.28 microm thickness), which is a fundamental determinant of the hydrodynamic and mechanical environment at the endothelial cell surface, is absent from human umbilical vein (0.03+/-0.04 microm thickness) and bovine aortic (0.02+/-0.04 microm thickness) endothelial cells grown and maintained under standard cell culture conditions in vitro. An endothelial surface-bound glycosaminoglycan layer, not necessarily indicative of but having similar hydrodynamic properties to the endothelial glycocalyx observed in vivo, was detected (0.21+/-0.27 microm thickness) only after hyaluronan and chondroitin sulfate were added to the cell culture media at hyperphysiological concentrations (0.2 mg/mL perfused for 75 minutes). The implications of this glycocalyx deficiency under standard cell culture conditions in these pervasive in vitro models broadly impact a myriad of studies involving endothelial cell monolayers in which inferences are made that may depend on endothelial cell surface chemistry. In light of these findings, conclusions drawn from such studies in the areas of microvascular permeability, inflammation, mechanotransduction, and atherosclerosis must be carefully reconsidered.

OBJECTIVE

To determine whether chondroitin sulfate (CS) is effective in inhibiting cartilage loss in knee osteoarthritis (OA).

METHODS

In this randomized, double-blind, placebo-controlled trial, 300 patients with knee OA were recruited from an outpatient clinic, from private practices, and through advertisements. Study patients were randomly assigned to receive either 800 mg CS or placebo once daily for 2 years. The primary outcome was joint space loss over 2 years as assessed by a posteroanterior radiograph of the knee in flexion; secondary outcomes included pain and function.

RESULTS

Of 341 patients screened, 300 entered the study and were included in the intent-to-treat analysis. The 150 patients receiving placebo had progressive joint space narrowing, with a mean +/- SD joint space loss of 0.14 +/- 0.61 mm after 2 years (P = 0.001 compared with baseline). In contrast, there was no change in mean joint space width for the 150 patients receiving CS (0.00 +/- 0.53 mm; P not significant compared with baseline). Similar results were found for minimum joint space narrowing. The differences in loss of joint space between the two groups were significant for mean joint space width (0.14 +/- 0.57 mm; P = 0.04) and for minimum joint space width (0.12 +/- 0.52 mm; P = 0.05). CS was well tolerated, with no significant differences in rates of adverse events between the two groups.

CONCLUSIONS

While there was no significant symptomatic effect in this study, long-term treatment with CS may retard radiographic progression in patients with OA of the knee. However, the clinical relevance of the observed structural results has to be further evaluated, and further studies are needed to confirm the structural effects of CS.

Chondroitin sulfate proteoglycans have been implicated in the regulation of cell migration and pattern formation in the developing peripheral nervous system. To identify whether the large aggregating proteoglycan versican might be mediating these processes, we prepared monospecific antibodies against a recombinant core protein fragment of chick versican. The purified antibodies recognize the predominant versican splice-variants V0 and V1. Using these antibodies, we revealed a close correlation between the spacio-temporal expression of versican and the formation of molecular boundaries flanking or transiently blocking the migration pathways of neural crest cells or motor and sensory axons. Versican is present in the caudal sclerotome, the early dorsolateral tissue underneath the ectoderm, the pelvic girdle precursor and to a certain extent in the perinotochordal mesenchyme. Versican is completely absent from tissues invaded by neural crest cells and extending axons. Upon completion of neural crest cell migration and axon outgrowth, versican expression is shifted to pre-chondrogenic areas. Since versican inhibits cellular interactions with fibronectin, laminin and collagen I in vitro, the selective expression of versican within barrier tissues may be linked to a functional role of versican in the guidance of migratory neural crest cells and outgrowing axons.

The human CD44 cell-surface glycoprotein participates in a wide variety of cell-cell interactions including lymphocyte homing and tumor metastasis. The CD44 antigen is known to display extensive size heterogeneity when compared between different tissue sources although the structural basis for this variation is not yet clear. Recently, two further isotypes in addition to the basic hemopoietic form of the CD44 antigen have been cloned and sequenced and these have been found to contain all or part of a 200-400-base pair insert within the extracellular domain, suggesting that the characteristic heterogeneity in the molecule may be generated by a mechanism of alternative splicing. We have obtained further evidence for alternative splicing, and we report here the cloning and sequencing of six different CD44 sequence variants from a variety of cell lines using a combination of expression cloning and the polymerase chain reaction. Comparison of these variants indicates that each is probably assembled by the insertion of five different exon units in tandem into a discrete site within the membrane proximal region of the extracellular domain. One of the variants contains an exon that shares extensive amino acid sequence homology with a recently described rat CD44 variant that mediates tumor metastasis. Another variant contains a new exon that encodes a tandem repeat of the consensus sequence SG for covalent modification with chondroitin sulfate and is expressed predominantly on mammary tumors. We suggest that a mechanism of alternative exon splicing generates much of the observed structural heterogeneity of CD44 and that the particular set of CD44 variants expressed in a single cell may represent a precise postal code directing the final destination of migrating cells and metastatic tumors.

We have identified the Drosophila UDP-glucose dehydrogenase gene as being involved in wingless signaling. Mutations in this gene, called kiwi, generate a phenotype identical to that of wingless. UDP-glucose dehydrogenase is required for the biosynthesis of UDP-glucuronate, which in turn is utilized in the biosynthesis of glycosaminoglycans. By rescuing the kiwi phenotype with both UDP-glucuronate and the glycosaminoglycan heparan sulfate, we show that kiwi function in the embryo is crucial for the production of heparan sulfate in the extracellular matrix. Further, injection of heparin degrading enzyme, heparinase (and not chondroitin, dermatan or hyaluronic acid degrading enzyme) into wild-type embryos leads to the degradation of heparin-like glycosaminoglycans and a 'wingless-like' cuticular phenotype. Our study thus provides the first genetic evidence for the involvement of heparin-like glycosaminoglycans in signal transduction.

OBJECTIVE

To determine the concentrations of human stromelysin-1, collagenase, tissue inhibitor of metalloproteinases (TIMP), and proteoglycan fragments in knee synovial fluid in patients with injury to the meniscus or anterior cruciate ligament, posttraumatic osteoarthritis, primary osteoarthritis, or pyrophosphate arthritis.

METHODS

Synovial fluid samples were collected from patients with knee disease diagnosed arthroscopically and radiologically. Concentrations of stromelysin-1, collagenase, and TIMP-1 were determined by sandwich immunoassay, using monoclonal and polyclonal antibodies. Fragments of cartilage proteoglycan containing the chondroitin sulfate-binding region were determined by immunoassay with a polyclonal antibody.

RESULTS

Average concentrations of metalloproteinases, TIMP, and proteoglycan fragments in joint fluid were significantly elevated in patients from all disease groups as compared with volunteers with healthy knees (reference group). Stromelysin concentrations in disease groups averaged 15-45 times that of the reference group. The molar ratios between stromelysin and collagenase varied between 10 and 150. The molar ratio between total stromelysin and free TIMP was 0.5 in the reference group and between 1.6 and 5.3 in the disease groups.

CONCLUSIONS

Stromelysin concentration in joint fluid is a parameter that distinguishes diseased joints from healthy joints, with a sensitivity of 84% and a specificity of 90%. The high concentrations of metalloproteinase relative to TIMP in joint fluid from patients with the conditions studied may be associated with cartilage matrix degradation in these arthritides.

The high molecular mass glycosaminoglycan hyaluronan (HA) can become modified by the covalent attachment of heavy chains (HCs) derived from the serum protein inter-alpha-inhibitor (IalphaI), which is composed of three subunits (HC1, HC2 and bikunin) linked together via a chondroitin sulfate moiety. The formation of HC.HA is likely to play an important role in the stabilization of HA-rich extracellular matrices in the context of inflammatory disease (e.g. arthritis) and ovulation. Here, we have characterized the complexes formed in vitro between purified human IalphaI and recombinant human TSG-6 (an inflammation-associated protein implicated previously in this process) and show that these complexes (i.e. TSG-6 x HC1 and TSG-6 x HC2) act as intermediates in the formation of HC x HA. This is likely to involve two transesterification reactions in which an ester bond linking an HC to chondroitin sulfate in intact IalphaI is transferred first onto TSG-6 and then onto HA. The formation of TSG-6 x HC1 and TSG-6 x C2 complexes was accompanied by the production of bikunin x HC2 and bikunin x HC1 by-products, respectively, which were observed to break down, releasing free bikunin and HCs. Both TSG-6 x HC formation and the subsequent HC transfer are metal ion-dependent processes; these reactions have a requirement for either Mg2+ or Mn2+ and are inhibited by Co2+. TSG-6, which is released upon the transfer of HCs from TSG-6 onto HA, was shown to combine with IalphaI to form new TSG-6 x HC complexes and thus be recycled. The finding that TSG-6 acts as cofactor and catalyst in the production of HC x HA complexes has important implications for our understanding of inflammatory and inflammation-like processes.

Cathepsin K, a lysosomal papain-like cysteine protease, forms collagenolytically highly active complexes with chondroitin sulfate and represents the most potent mammalian collagenase. Here we demonstrate that complex formation with glycosaminoglycans (GAGs) is unique for cathepsin K among human papain-like cysteine proteases and that different GAGs compete for the binding to cathepsin K. GAGs predominantly expressed in bone and cartilage, such as chondroitin and keratan sulfates, enhance the collagenolytic activity of cathepsin K, whereas dermatan, heparan sulfate, and heparin selectively inhibit this activity. Moreover, GAGs potently inhibit the collagenase activity of other cysteine proteases such as cathepsins L and S at 37 degrees C. Along this line MMP1-generated collagen fragments in the presence of GAGs are stable against further degradation at 28 degrees C by all cathepsins but cathepsin K, whereas thermal destabilization at 37 degrees C renders the fragments accessible to all cathepsins. These results suggest a novel mechanism for the regulation of matrix protein degradation by GAGs. It further implies that cathepsin K represents the only lysosomal collagenolytic activity under physiologically relevant conditions.