Neurotropin® ameliorates chronic pain associated with scar formation in a mouse model: A gene expression analysis of the inflammatory response.

Background: Scar formation after trauma and surgery involves an inflammatory response and can lead to the development of chronic pain. Neurotropin® (NTP) is a nonprotein extract of inflamed skin of rabbits inoculated with vaccinia virus. It has been widely used for the treatment of chronic pain. However, the in vivo effects of NTP on painful scar formation have not been determined. To investigate the molecular mechanisms underlying the effects of NTP on the inflammatory response, we evaluated gene expression in the scar tissues and dorsal root ganglions (DRGs) of mice administered NTP and control mice. Methods and results: Mice injected with saline or NTP were used as controls; other mice were subjected to surgery on the left hind paw to induce painful scar formation, and then injected with saline or NTP. Hind paw pain was evaluated by measuring the threshold for mechanical stimulation using the von Frey test. The paw withdrawal threshold gradually returned to pre-operative levels over 4 weeks post-operation; NTP-treated mice showed a significantly shortened recovery time of approximately 3 weeks, suggesting that NTP exerted an analgesic effect in this mouse model. Total RNA was extracted from the scarred hind paw tissues and DRGs were collected 1 week post-operation for a microarray analysis. Gene set enrichment analysis revealed that the expression of some gene sets related to inflammatory responses was activated or inhibited following surgery and NTP administration. Quantitative real-time reverse transcription-polymerase chain reaction analysis results for several genes were consistent with the microarray results. Conclusion: The administration of NTP to the hind paws of mice with painful scar formation following surgery diminished nociceptive pain and reduced the inflammatory response. NTP inhibited the expression of some genes involved in the response to surgery-induced inflammation. Therefore, NTP is a potential therapeutic option for painful scar associated with chronic pain.

Possible Repositioning of an Oral Anti-Osteoporotic Drug, Ipriflavone, for Treatment of Inflammatory Arthritis via Inhibitory Activity of KIAA1199, a Novel Potent Hyaluronidase.

KIAA1199 has a strong hyaluronidase activity in inflammatory arthritis. This study aimed to identify a drug that could reduce KIAA1199 activity and clarify its effects on inflammatory arthritis. Rat chondrosarcoma (RCS) cells were strongly stained with Alcian blue (AB). Its stainability was reduced in RCS cells, which were over-expressed with the KIAA1199 gene (RCS-KIAA). We screened the drugs that restore the AB stainability in RCS-KIAA. The effects of the drug were evaluated by particle exclusion assay, HA ELISA, RT-PCR, and Western blotting. We further evaluated the HA accumulation and the MMP1 and three expressions in fibroblast-like synoviocytes (FLS). In vivo, the effects of the drug on symptoms and serum concentration of HA in a collagen-induced arthritis mouse were evaluated. Ipriflavone was identified to restore AB stainability at 23%. Extracellular matrix formation was significantly increased in a dose-dependent manner (p = 0.006). Ipriflavone increased the HA accumulation and suppressed the MMP1 and MMP3 expression on TNF-α stimulated FLS. In vivo, Ipriflavone significantly improved the symptoms and reduced the serum concentrations of HA. Conclusions: We identified Ipriflavone, which has inhibitory effects on KIAA1199 activity. Ipriflavone may be a therapeutic candidate based on its reduction of KIAA1199 activity in inflammatory arthritis.

Forced expression of KIAA1199, a novel hyaluronidase, inhibits tumorigenicity of low-grade chondrosarcoma.

Hyaluronan (HA) has been shown to play crucial roles in the tumorigenicity of malignant tumors. Chondrosarcoma, particularly when low-grade, is characterized by the formation of an extracellular matrix (ECM) containing abundant HA, and its drug/radiation resistance has become a clinically relevant problem. This study aimed to evaluate the effects of a novel hyaluronidase, KIAA1199, on ECM formation as well as antitumor effects on chondrosarcoma. To clarify the roles of KIAA1199 in chondrosarcoma, mouse KIAA1199 was stably transfected to Swarm rat chondrosarcoma (RCS) cells (histologically grade 1). We investigated the effects of KIAA1199 on RCS cells in vitro and an autografted model in vivo. HA binding protein (HABP) stainability and ECM formation in KIAA1199-RCS was markedly suppressed compared with that of control cells. No significant changes in messenger RNA expression of Has1, Has2, Has3, Hyal1, or Hyal2 were observed. KIAA1199 expression did not affect proliferation or apoptosis but inhibited migration and invasion of RCS cells. In contrast, the expression of KIAA1199 significantly inhibited the growth of grafted tumors and suppressed the stainability of alcian blue in tumor tissues. Although there was no direct inhibitory effect on proliferation in vitro, induction of KIAA1199 showed the antitumor effects in grafted tumor growth in vivo possibly due to changes in the tumor microenvironment such as inhibition of ECM formation. Forced expression of KIAA1199 exhibits antitumor effects on low-grade chondrosarcoma, which has chemo- and radio-therapy resistant features. Together, KIAA1199 could be a novel promising therapeutic tool for low-grade chondrosarcoma, mediated by the degradation of HA.

Suppression of hyaluronan synthesis attenuates the tumorigenicity of low-grade chondrosarcoma.

Hyaluronan (HA) has been shown to play crucial roles in the tumorigenicity of malignant tumors. Chondrosarcoma, particularly when low-grade, is characterized by the formation of an extracellular matrix (ECM) containing abundant HA, and its drug/radiation resistance has become a clinically relevant problem. This study aimed to evaluate the effects of an HA synthesis inhibitor, 4-methylumbelliferone (MU), on ECM formation as well as antitumor effects in chondrosarcoma. We investigated the effects of MU on rat chondrosarcoma (RCS) cells with a grade I histological malignancy in vitro and in vivo grafted model. HA binding protein (HABP) stainability on and around the RCS cells was effectively reduced with treatment of MU. ECM formation was markedly suppressed by MU at a dose of 1.0 mM. Cell proliferation was significantly reduced by MU at 24 h. Cell motility and invasion were suppressed in a dose-dependent manner by MU. No significant changes in mRNA expression of Has1-3 were observed. Furthermore, MU inhibited the growth of grafted tumors in vivo. Histologically, chondrosarcoma cells of control tumors showed a cell-clustering structure. HABP stainability was markedly decreased in the MU-treated group. These results suggest that MU exhibits antitumor effects on low-grade chondrosarcoma, via inhibition of HA accumulation and ECM formation. MU, which is an approved drug in bile therapy, could be a new off-label medication for chondrosarcomas. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1573-1580, 2018.

Postnatal lethality and chondrodysplasia in mice lacking both chondroitin sulfate N-acetylgalactosaminyltransferase-1 and -2.

Chondroitin sulfate (CS) is a sulfated glycosaminoglycan (GAG) chain. In cartilage, CS plays important roles as the main component of the extracellular matrix (ECM), existing as side chains of the major cartilage proteoglycan, aggrecan. Six glycosyltransferases are known to coordinately synthesize the backbone structure of CS; however, their in vivo synthetic mechanism remains unknown. Previous studies have suggested that two glycosyltransferases, Csgalnact1 (t1) and Csgalnact2 (t2), are critical for initiation of CS synthesis in vitro. Indeed, t1 single knockout mice (t1 KO) exhibit slight dwarfism and a reduction in CS content in cartilage compared with wild-type (WT) mice. To reveal the synergetic roles of t1 and t2 in CS synthesis in vivo, we generated systemic single and double knockout (DKO) mice and cartilage-specific t1 and t2 double knockout (Col2-DKO) mice. DKO mice exhibited postnatal lethality, whereas t2 KO mice showed normal size and skeletal development. Col2-DKO mice survived to adulthood and showed severe dwarfism compared with t1 KO mice. Histological analysis of epiphyseal cartilage from Col2-DKO mice revealed disrupted endochondral ossification, characterized by drastic GAG reduction in the ECM. Moreover, DKO cartilage had reduced chondrocyte proliferation and an increased number of apoptotic chondrocytes compared with WT cartilage. Conversely, primary chondrocyte cultures from Col2-DKO knee cartilage had the same proliferation rate as WT chondrocytes and low GAG expression levels, indicating that the chondrocytes themselves had an intact proliferative ability. Quantitative RT-PCR analysis of E18.5 cartilage showed that the expression levels of Col2a1 and Ptch1 transcripts tended to decrease in DKO compared with those in WT mice. The CS content in DKO cartilage was decreased compared with that in t1 KO cartilage but was not completely absent. These results suggest that aberrant ECM caused by CS reduction disrupted endochondral ossification. Overall, we propose that both t1 and t2 are necessary for CS synthesis and normal chondrocyte differentiation but are not sufficient for all CS synthesis in cartilage.

Serum-Derived Hyaluronan-Associated Protein Is a Novel Biomarker for Inflammatory Bowel Diseases.

BACKGROUND/AIMS: We evaluated the role of serum-derived hyaluronan-associated protein (SHAP) in inflammatory bowel disease (IBD) pathogenesis and its potential as a novel IBD biomarker. METHODS: We studied the SHAP expression in a mouse model of colitis and in human intestinal samples of IBD and compared serum concentrations with normal controls. RESULTS: SHAP was expressed in the connective tissue derived from inflamed regions of the intestine. In mice, serum levels of SHAP-hyaluronic acid (SHAP-HA) were positively correlated with the histological damage of the colon (r = 0.566, p < 0.001). Serum concentration of SHAP-HA complex was significantly higher in patients with active ulcerative colitis than in those in remission, and this value was positively correlated with the erythrocyte sedimentation rate, serum level of tumor necrosis factor (TNF)-α, and endoscopic damage (r = 0.568, p < 0.001; r = 0.521, p < 0.001, and r = 0.641, p < 0.001). In patients with Crohn's disease, the serum SHAP-HA level correlated only with TNF-α (r = 0.630, p = 0.002). CONCLUSION: SHAP is a novel IBD biomarker that is related to disease activity in certain types of colitis, and it may affect disease pathogenesis. Future studies are needed to evaluate the therapeutic potential of this complex.

Antitumor effects of 4-methylumbelliferone, a hyaluronan synthesis inhibitor, on malignant peripheral nerve sheath tumor.

Hyaluronan (HA) has been shown to play important roles in the growth, invasion and metastasis of malignant tumors. Our previous study showing that high HA expression in malignant peripheral nerve sheath tumors (MPNST) is predictive of poor patient prognosis, prompted us to speculate that inhibition of HA synthesis in MPNST might suppress the tumorigenicity. The aim of our study was to investigate the antitumor effects of 4-methylumbelliferone (MU), an HA synthesis inhibitor, on human MPNST cells and tissues. The effects of MU on HA accumulation and tumorigenicity in MPNST cells were analyzed in the presence or absence of MU in an in vitro as well as in vivo xenograft model using human MPNST cell lines, sNF96.2 (primary recurrent) and sNF02.2 (metastatic). MU significantly inhibited cell proliferation, migration and invasion in both MPNST cell lines. HA binding protein (HABP) staining, particle exclusion assay and quantification of HA revealed that MU significantly decreased HA accumulation in the cytoplasms and pericellular matrices in both MPNST cell lines. The expression levels of HA synthase2 (HAS2) and HA synthase3 (HAS3) mRNA were downregulated after treatment with MU. MU induced apoptosis of sNF96.2 cells, but not sNF02.2 cells. MU administration significantly inhibited the tumor growth of sNF96.2 cells in the mouse xenograft model. To the best of our knowledge, our study demonstrates for the first time the antitumor effects of MU on human MPNST mediated by inhibition of HA synthesis. Our results suggest that MU may be a promising agent with novel antitumor mechanisms for MPNST.

Large-scale mutational analysis in the EXT1 and EXT2 genes for Japanese patients with multiple osteochondromas.

BACKGROUND: Multiple osteochondroma (MO) is an autosomal dominant skeletal disorder characterized by the formation of multiple osteochondromas, and exostosin-1 (EXT1) and exostosin-2 (EXT2) are major causative genes in MO. In this study, we evaluated the genetic backgrounds and mutational patterns in Japanese families with MO. RESULTS: We evaluated 112 patients in 71 families with MO. Genomic DNA was isolated from peripheral blood leucocytes. The exons and exon/intron junctions of EXT1 and EXT2 were directly sequenced after PCR amplification. Fifty-two mutations in 47 families with MO in either EXT1 or EXT2, and 42.3% (22/52) of mutations were novel mutations. Twenty-nine families (40.8%) had mutations in EXT1, and 15 families (21.1%) had mutations in EXT2. Interestingly, three families (4.2%) had mutations in both EXT1 and EXT2. Twenty-four families (33.8%) did not exhibit mutations in either EXT1 or EXT2. With regard to the types of mutations identified, 59.6% of mutations were inactivating mutations, and 38.5% of mutations were missense mutations. CONCLUSIONS: We found that the prevalence of EXT1 mutations was greater than that of EXT2 mutations in Japanese MO families. Additionally, we identified 22 novel EXT1 and EXT2 mutations in this Japanese MO cohort. This study represents the variety of genotype in MO.

Key Roles of Hyaluronan and Its CD44 Receptor in the Stemness and Survival of Cancer Stem Cells.

Cancer stem cells (CSCs) represent a unique subpopulation of self-renewing oncogenic cells that drive cancer initiation and progression. CSCs often acquire multidrug and oxidative stress resistance and are thereby thought to be responsible for tumor recurrence following treatment and remission. Although the mechanisms responsible for CSC generation, maintenance, and expansion have become a major focus in cancer research, the molecular characteristics of CSCs remain poorly understood. The stemness and subsequent expansion of CSCs are believed to be highly influenced by changes in microenvironmental signals as well as genetic and epigenetic alterations. Hyaluronan (HA), a major component of the extracellular matrix, has recently been demonstrated to provide a favorable microenvironment for the self-renewal and maintenance of stem cells. HA directly and indirectly affects CSC self-renewal by influencing the behavior of both cancer and stromal cells. For instance, HA in the tumor microenvironment modulates the function of tumor-associated macrophages to support CSC self-renewal, and excessive HA production promotes the acquisition of CSC signatures through epithelial-to-mesenchymal transition. The importance of HA in mediating CSC self-renewal has been strengthened by the finding that interactions between HA and its receptor, CD44, propagate the stemness of CSCs. HA-CD44 interactions evoke a wide range of signals required for CSC self-renewal and maintenance. CD44 also plays a critical role in the preservation and multidrug resistance (MDR) of CSCs by transmitting survival and anti-apoptotic signals. Thus, a better understanding of the molecular mechanisms involved in HA and CD44 control of CSC stemness may help in the design of more effective therapies for cancer patients. In this review, we address the key roles of HA and CD44 in CSC self-renewal and maintenance. We also discuss the involvement of CD44 in the oxidative stress and MDR of CSCs.

Acute and temporal expression of tumor necrosis factor (TNF)-α-stimulated gene 6 product, TSG6, in mesenchymal stem cells creates microenvironments required for their successful transplantation into muscle tissue.

Previously, we demonstrated that when mesenchymal stem cells (MSCs) from mouse ES cells were transplanted into skeletal muscle, more than 60% of them differentiated into muscles in the crush-injured tibialis anterior muscle in vivo, although MSCs neither differentiated nor settled in the intact muscle. Microenvironments, including the extracellular matrix between the injured and intact muscle, were quite different. In the injured muscle, hyaluronan (HA), heavy chains of inter-α-inhibitor (IαI), CD44, and TNF-α-stimulated gene 6 product (TSG-6) increased 24-48 h after injury, although basement membrane components of differentiated muscle such as perlecan, laminin, and type IV collagen increased gradually 4 days after the crush. We then investigated the microenvironments crucial for cell transplantation, using the lysate of C2C12 myotubules for mimicking injured circumstances in vivo. MSCs settled in the intact muscle when they were transplanted together with the C2C12 lysate or TSG6. MSCs produced and released TSG6 when they were cultured with C2C12 lysates in vitro. MSCs pretreated with the lysate also settled in the intact muscle. Furthermore, MSCs whose TSG6 was knocked down by shRNA, even if transplanted or pretreated with the lysate, could not settle in the muscle. Immunofluorescent staining showed that HA and IαI always co-localized or were distributed closely, suggesting formation of covalent complexes, i.e. the SHAP-HA complex in the presence of TSG6. Thus, TSG6, HA, and IαI were crucial factors for the settlement and probably the subsequent differentiation of MSCs.

Sulfated glycosaminoglycans control the extracellular trafficking and the activity of the metalloprotease inhibitor TIMP-3.

Tissue inhibitor of metalloproteinase 3 (TIMP-3) is an important regulator of extracellular matrix (ECM) turnover. TIMP-3 binds to sulfated ECM glycosaminoglycans or is endocytosed by cells via low-density lipoprotein receptor-related protein 1 (LRP-1). Here, we report that heparan sulfate (HS) and chondroitin sulfate E (CSE) selectively regulate postsecretory trafficking of TIMP-3 by inhibiting its binding to LRP-1. HS and CSE also increased TIMP-3 affinity for glycan-binding metalloproteinases, such as adamalysin-like metalloproteinase with thrombospondin motifs 5 (ADAMTS-5), by reducing the dissociation rate constants. The sulfation pattern was crucial for these activities because monosulfated or truncated heparin had a reduced ability to bind to TIMP-3 and increase its affinity for ADAMTS-5. Therefore, sulfation of ECM glycans regulates the levels and inhibitory activity of TIMP-3 and modulates ECM turnover, and small mimicries of sulfated glycans may protect the tissue from the excess destruction seen in diseases such as osteoarthritis, cancer, and atherosclerosis.

Hyaluronan expression as a significant prognostic factor in patients with malignant peripheral nerve sheath tumors.

Hyaluronan (HA) regulates malignant tumor growth, invasion, and metastasis. However, few studies have focused on the roles of HA in tumorigenicity in malignant peripheral nerve sheath tumors (MPNST). In this study, we sought to clarify the prognostic value of HA in patients with MPNST. Specimens obtained from 15 patients with neurofibroma and 30 with MPNST were subjected to HA staining and scored as three grades. Protein expressions of HA synthase 1-3 were examined in the 22 MPNST tissue samples available. Statistically higher HA positivity was observed in MPNST as compared with neurofibroma (P = 0.020). The univariate analysis revealed that increased HA expression, age, neurofibromatosis type 1 (NF1) status, large tumor size, and histological grade were significantly associated with reduced overall survival of patients with MPNST; while increased HA expression, NF1 status, tumor size, and histological grade were correlated with disease-free survival. However, HA synthase 1-3 expression related to neither overall survival nor disease-free survival of these patients. In multivariate analysis, large tumor size (P = 0.022) was an independent prognostic factor for overall survival, and HA expression (P = 0.028) and tumor size (P = 0.002) were independent prognostic factors for disease-free survival. Statistically higher levels of HA in the human MPNST cells were observed compared with neurofibroma cells in vitro. Our results demonstrate that HA expression can be a useful marker in differentiating MPNST from neurofibroma, and in identifying patients with a poor prognosis. Hyaluronan-targeting therapy for patients with MPNST may have potential as a therapeutic tool.

Hyaluronan deficiency due to Has3 knock-out causes altered neuronal activity and seizures via reduction in brain extracellular space.

Hyaluronan (HA), a large anionic polysaccharide (glycosaminoglycan), is a major constituent of the extracellular matrix of the adult brain. To address its function, we examined the neurophysiology of knock-out mice deficient in hyaluronan synthase (Has) genes. Here we report that these Has mutant mice are prone to epileptic seizures, and that in Has3(-/-) mice, this phenotype is likely derived from a reduction in the size of the brain extracellular space (ECS). Among the three Has knock-out models, namely Has3(-/-), Has1(-/-), and Has2(CKO), the seizures were most prevalent in Has3(-/-) mice, which also showed the greatest HA reduction in the hippocampus. Electrophysiology in Has3(-/-) brain slices demonstrated spontaneous epileptiform activity in CA1 pyramidal neurons, while histological analysis revealed an increase in cell packing in the CA1 stratum pyramidale. Imaging of the diffusion of a fluorescent marker revealed that the transit of molecules through the ECS of this layer was reduced. Quantitative analysis of ECS by the real-time iontophoretic method demonstrated that ECS volume was selectively reduced in the stratum pyramidale by ∼ 40% in Has3(-/-) mice. Finally, osmotic manipulation experiments in brain slices from Has3(-/-) and wild-type mice provided evidence for a causal link between ECS volume and epileptiform activity. Our results provide the first direct evidence for the physiological role of HA in the regulation of ECS volume, and suggest that HA-based preservation of ECS volume may offer a novel avenue for development of antiepileptogenic treatments.

Homotypic versican G1 domain interactions enhance hyaluronan incorporation into fibrillin microfibrils.

Versican G1 domain-containing fragments (VG1Fs) have been identified in extracts from the dermis in which hyaluronan (HA)-versican-fibrillin complexes are found. However, the molecular assembly of VG1Fs in the HA-versican-microfibril macrocomplex has not yet been elucidated. Here, we clarify the role of VG1Fs in the extracellular macrocomplex, specifically in mediating the recruitment of HA to microfibrils. Sequential extraction studies suggested that the VG1Fs were not associated with dermal elements through HA binding properties alone. Overlay analyses of dermal tissue sections using the recombinant versican G1 domain, rVN, showed that rVN deposited onto the elastic fiber network. In solid-phase binding assays, rVN bound to isolated nondegraded microfibrils. rVN specifically bound to authentic versican core protein produced by dermal fibroblasts. Furthermore, rVN bound to VG1Fs extracted from the dermis and to nondenatured versican but not to fibrillin-1. Homotypic binding of rVN was also seen. Consistent with these binding properties, macroaggregates containing VG1Fs were detected in high molecular weight fractions of sieved dermal extracts and visualized by electron microscopy, which revealed localization to microfibrils at the microscopic level. Importantly, exogenous rVN enhanced HA recruitment both to isolated microfibrils and to microfibrils in tissue sections in a dose-dependent manner. From these data, we propose that cleaved VG1Fs can be recaptured by microfibrils through VG1F homotypical interactions to enhance HA recruitment to microfibrils.

Glycosaminoglycans in the blood of hereditary multiple exostoses patients: Half reduction of heparan sulfate to chondroitin sulfate ratio and the possible diagnostic application.

Hereditary multiple exostoses (HME) is an autosomal dominant skeletal disorder with wide variation in clinical phenotype and is caused by heterogeneous germline mutations in two of the Ext genes, EXT-1 and EXT-2, which encode ubiquitously expressed glycosyltransferases involved in the polymerization of heparan sulfate (HS) chains. To examine whether the Ext mutation could affect HS structures and amounts in HME patients being heterozygous for the Ext genes, we collected blood from patients and healthy individuals, separated it into plasma and cellular fractions and then isolated glycosaminoglycans (GAGs) from those fractions. A newly established method consisting of a combination of selective ethanol precipitation of GAGs, digestion of GAGs recovered on the filter-cup by direct addition of heparitinase or chondroitinase reaction solution and subsequent high-performance liquid chromatography of the unsaturated disaccharide products enabled the analysis using the least amount of blood (200 µL). We found that HS structures of HME patients were almost similar to those of controls in both plasma and cellular fractions. However, interestingly, although both the amounts of HS and chondroitin sulfate (CS) varied depending on the different individuals, the amounts of HS in both the plasma and cellular fractions of HME patient samples were decreased and the ratios of HS to CS (HS/CS) of HME patient samples were almost half those of healthy individuals. The results suggest that HME patients' blood exhibited reduced HS amounts and HS/CS ratios, which could be used as a diagnostic biomarker for HME.

Suppression of hyaluronan synthesis alleviates inflammatory responses in murine arthritis and in human rheumatoid synovial fibroblasts.

OBJECTIVE: To clarify the roles of hyaluronan (HA) in joint inflammation and the process of joint destruction, using 4-methylumbelliferone (4-MU), an inhibitor of HA synthesis, in a mouse model of collagen-induced arthritis (CIA) and in a monolayer culture of fibroblast-like synoviocytes (FLS) derived from patients with rheumatoid arthritis. METHODS: DAB/1J mice were immunized with type II collagen. The effects of 4-MU were evaluated by the physiologic arthritis score, paw swelling, the histologic arthritis score, and expression of matrix metalloproteinase 3 (MMP-3) and MMP-13 in chondrocytes and synovial tissue. In vitro, the effect of 4-MU on messenger RNA and protein expression of MMP-1 and MMP-3 was determined. The effects of 4-MU on HA deposition and on serum/medium concentrations of HA were analyzed using biotinylated HA binding protein staining and an HA binding assay, respectively. RESULTS: Treatment with 4-MU in mice with CIA dramatically decreased the severity of arthritis (based on the arthritis score), paw thickness, and histopathologic changes. MMP-3 and MMP-13 expression in chondrocytes and synovial cells was significantly inhibited by 4-MU in vivo. Treatment with 4-MU also inhibited MMP-1 and MMP-3 expression in tumor necrosis factor α-stimulated FLS, in a dose-dependent manner. The 4-MU-induced decreases in the serum HA concentration in mice with CIA and in "medium" and "pericellular" HA concentrations in cultured FLS support the contention that the inhibitory mechanism of 4-MU is mediated by HA suppression. CONCLUSION: Reduced disease activity induced by 4-MU in mice with CIA revealed HA to be a crucial regulator in the course of arthritis. Therefore, 4-MU is a potential therapeutic agent in arthritis, and its inhibitory mechanism is possibly mediated by suppression of HA synthesis.

The crystal structure of novel chondroitin lyase ODV-E66, a baculovirus envelope protein.

Chondroitin lyases have been known as pathogenic bacterial enzymes that degrade chondroitin. Recently, baculovirus envelope protein ODV-E66 was identified as the first reported viral chondroitin lyase. ODV-E66 has low sequence identity with bacterial lyases at <12%, and unique characteristics reflecting the life cycle of baculovirus. To understand ODV-E66's structural basis, the crystal structure was determined and it was found that the structural fold resembled that of polysaccharide lyase 8 proteins and that the catalytic residues were also conserved. This structure enabled discussion of the unique substrate specificity and the stability of ODV-E66 as well as the host specificity of baculovirus.

Inhibitory effect of chondroitin sulfate type E on the binding step of human T-cell leukemia virus type 1.

Cell surface heparan sulfate proteoglycans (HSPGs) are involved in the binding and entry of human T-cell leukemia virus type 1 (HTLV-1) into host cells, while sulfated polysaccharides such as heparin inhibit HTLV-1 infection. Chondroitin sulfate proteoglycans (CSPGs) are classified as another major type of proteoglycans. Here, we examined the effect of four types of chondroitin sulfate (CS) on HTLV-1 infection. Accordingly, a human T cell line, MOLT-4, was inoculated with cell-free HTLV-1 in the presence or absence of soluble CS, and the synthesis of reverse-transcribed HTLV-1 DNA within cells 20 h after inoculation was detected using polymerase chain reaction (PCR). Among the four types of CS (A, C, D, and E), the E type (CSE), which was derived from the squid cartilage, exhibited anti-HTLV-1 activity. Furthermore, we observed that CSE directly interacted with recombinant HTLV-1 envelope (Env) proteins and inhibited the binding of HTLV-1 virions to MOLT-4 cells, indicating that the interaction between Env and CSE plays a significant role in its anti-HTLV-1 activity. In addition, CSE inhibited syncytium formation that was induced by HTLV-1-producing cells. When CSE was mixed with the synthetic fusion inhibitor peptide corresponding to the ectodomain of the Env transmembrane subunit (TM) gp21, the HTLV-1 infection was further inhibited when compared with the inhibitory effect of each compound alone. Thus, further elucidation of the in vitro antiviral mechanism of CSE shown in this study will lead to the development of CSE-like molecules for the entry inhibition of HTLV-1.

The structural basis for a coordinated reaction catalyzed by a bifunctional glycosyltransferase in chondroitin biosynthesis.

Bifunctional chondroitin synthase K4CP catalyzes glucuronic acid and N-acetylgalactosamine transfer activities and polymerizes a chondroitin chain. Here we have determined that an N-terminal region (residues 58-134) coordinates two transfer reactions and enables K4CP to catalyze polymerization. When residues 58-107 are deleted, K4CP loses polymerase activity while retaining both transfer activities. Peptide (113)DWPSDL(118) within this N-terminal region interacts with C-terminal peptide (677)YTWEKI(682). The deletion of either sequence abolishes glucuronic acid but not N-acetylgalactosamine transfer activity in K4CP. Both donor bindings and transfer activities are lost by mutating (677)YTWEKI(682) to (677)DAWEDI(682). On the other hand, acceptor substrates retain their binding to K4CP mutants. The characteristics of these K4CP mutants highlight different states of the enzyme reaction, providing an underlying structural basis for how these peptides play essential roles in coordinating the two glycosyltransferase activities for K4CP to elongate the chondroitin chain.

Hyaluronan expressed by the hematopoietic microenvironment is required for bone marrow hematopoiesis.

The contribution of hyaluronan (HA) to the regulatory network of the hematopoietic microenvironment was studied using knock-out mice of three hyaluronan synthase genes (Has1, Has2, and Has3). The number of hematopoietic progenitors was decreased in bone marrow and increased in extramedullary sites of Prx1-Cre;Has2(flox/flox);Has1(-/-);Has3(-/-) triple knock-out (tKO) mice as compared with wild type (WT) and Has1(-/-);Has3(-/-) double knock-out (dKO) mice. In line with this observation, decreased hematopoietic activity was observed in long term bone marrow cultures (LTBMC) from tKO mice, whereas the formation of the adherent layer and generation of hematopoietic cells in WT and dKO cultures was not different. 4-Methylumbelliferone (4MU) was used to pharmacologically inhibit the production of HA in LTBMC. Treatment with 4MU inhibited HA synthesis, decreased expression of HAS2 and HAS3, and eliminated hematopoiesis in LTBMC, and this effect was alleviated by the addition of exogenous HA. Exogenous HA also augmented the cell motility in LTBMC, which correlated with the HA-stimulated production of chemokines and growth factors. Conditioned media from HA-induced LTBMC enhanced the chemotaxis of hematopoietic stem/progenitor cells (HSPC) in response to SDF-1. Exposure of endothelial cells to 4MU decreased their ability to support HSPC rolling and adhesion. In addition, migration of transplanted HSPC into the marrow of 4MU-pretreated mice was lower than in untreated mice. Collectively, the results suggest that HA depletion reduces the ability of the microenvironment to support HSPC, and confirm a role for HA as a necessary regulatory element in the structure of the hematopoietic microenvironment.