Versican maintains the homeostasis of adipose tissues and regulates energy metabolism.

Versican is a large chondroitin sulfate proteoglycan in the extracellular matrix. It plays a pivotal role in the formation of the provisional matrix. S100a4, previously known as fibroblast-specific protein, functions as a calcium channel-binding protein. To investigate the role of versican expressed in fibroblasts, we generated conditional knockout mice in which versican expression is deleted in cells expressing S100a4. We found that S100a4 is expressed in adipose tissues, and these mice exhibit obesity under a normal diet, which becomes apparent as early as five months. The white adipose tissues of these mice exhibited decreased expression levels of S100a4 and versican and hypertrophy of adipocytes. qRT-PCR showed a reduced level of UCP1 in their white adipose tissues, indicating that the basic energy metabolism is diminished. These results suggest that versican in adipose tissues maintains the homeostasis of adipose tissues and regulates energy metabolism.

Aggrecan and versican: two brothers close or apart.

Aggrecan (Acan) and versican (Vcan) are large chondroitin sulfate proteoglycans of the extracellular matrix. They share the same structural domains at both N- and C-termini. The N-terminal G1 domain binds hyaluronan (HA), forms an HA-rich matrix, and regulates HA-mediated signaling. The C-terminal G3 domain binds other extracellular matrix molecules and forms a supramolecular structure that stores transforming growth factor ß (TGFß) and bone morphogenetic proteins (BMPs) and regulates their signaling. EGF-like motifs in the G3 domain may directly act like an EGF ligand. Both Acan and Vcan are present in cartilage, intervertebral disc, brain, heart, and aorta. Their localizations are essentially reciprocal. This review describes their structural domains, expression patterns and functions, and regulation of their expression.

Frontotemporal EEG as potential biomarker for early MCI: a case-control study.

BACKGROUND: Previous studies using EEG (electroencephalography) as biomarker for dementia have attempted to research, but results have been inconsistent. Most of the studies have extremely small number of samples (average N = 15) and studies with large number of data do not have control group. We identified EEG features that may be biomarkers for dementia with 120 subjects (dementia 10, MCI 33, against control 77). METHODS: We recorded EEG from 120 patients with dementia as they stayed in relaxed state using a single-channel EEG device while conducting real-time noise reduction and compared them to healthy subjects. Differences in EEG between patients and controls, as well as differences in patients' severity, were examined using the ratio of power spectrum at each frequency. RESULTS: In comparing healthy controls and dementia patients, significant power spectrum differences were observed at 3 Hz, 4 Hz, and 10 Hz and higher frequencies. In patient group, differences in the power spectrum were observed between asymptomatic patients and healthy individuals, and between patients of each respective severity level and healthy individuals. CONCLUSIONS: A study with a larger sample size should be conducted to gauge reproducibility, but the results implied the effectiveness of EEG in clinical practice as a biomarker of MCI (mild cognitive impairment) and/or dementia.

Characterization and functional analysis of novel circulating NK cell sub-populations.

Natural killer (NK) cells are innate lymphoid cells having potent cytolytic function that provide host defense against microbial infections and tumors. Using our generated monoclonal antibody (mAb), named FE-1H10, new NK cell sub-populations in peripheral blood were identified. The molecules recognized by mAb FE-1H10 were expressed on a sub-population of CD3-CD56dim NK cells. The epitope recognized by mAb FE-1H10 was demonstrated to be N-glycan and proven to be different from CD57. Upon K562 stimulation, the CD56dimFE-1H10+ NK cell sub-population exhibited significantly lower cytolytic function with low ability to degranulate and release cytolytic granules compared to the CD56dimFE-1H10- NK cell sub-population. Moreover, the CD56dimFE-1H10+ NK cells produced less IFN-γ and TNF-α than the CD56dimFE-1H10- NK cells. We demonstrated here that mAb FE-1H10 could identify two sub-populations of circulating CD56dim NK cells with different functions. Our discovery of new sub-populations of NK cells improves our understanding of NK cell biology and may lead to the development of new approaches for NK cell therapy.

Proteoglycan Chemical Diversity Drives Multifunctional Cell Regulation and Therapeutics.

The extracellular matrix (ECM) constitutes a highly dynamic three-dimensional structural network comprised of macromolecules, such as proteoglycans/glycosaminoglycans (PGs/GAGs), collagens, laminins, fibronectin, elastin, other glycoproteins and proteinases. In recent years, the field of PGs has expanded rapidly. Due to their high structural complexity and heterogeneity, PGs mediate several homeostatic and pathological processes. PGs consist of a protein core and one or more covalently attached GAG chains, which provide the protein cores with the ability to interact with several proteins. The GAG building blocks of PGs significantly influence the chemical and functional properties of PGs. The primary goal of this comprehensive review is to summarize major achievements and paradigm-shifting discoveries made on the PG/GAG chemistry-biology axis, focusing on structural variability, structure-function relationships, metabolic, molecular, and epigenetic mechanisms underlying their synthesis. Recent insights related to exosome biogenesis, degradation, and cell signaling, their status as diagnostic tools and potential pharmacological targets in diseases as well as current applications in nanotechnology and biotechnology are addressed. Moreover, issues related to docking studies, molecular modeling, GAG/PG interaction networks, and their integration are discussed.

The plant alkaloid conophylline inhibits matrix formation of fibroblasts.

Conophylline is a Vinca alkaloid from leaves of the tropical plant Ervatamia microphylla and has been shown to mimic the effect of the growth and differentiation factor activin A on pancreatic progenitor cells. However, activin A stimulates fibrosis of pancreatic stellate cells, whereas conophylline inhibits it, suggesting that this compound may serve as an antifibrotic drug. Here we investigated the effects of conophylline on human foreskin fibroblasts, especially focusing on extracellular matrix (ECM) proteins. A gene microarray analysis revealed that conophylline remarkably suppressed expression of the gene for hyaluronan synthase 2 (HAS2) and of its antisense RNA, whereas the expression of collagen genes was unaffected. Of note, immunostaining experiments revealed that conophylline substantially inhibits incorporation of versican and collagens into the ECM in cells treated with transforming growth factor ß (TGFß), which promotes collagen synthesis, but not in cells not treated with TGFß. Moreover, a protein biosynthesis assay disclosed that conophylline decreases collagen biosynthesis, concomitant with a decrease in total protein biosynthesis, indicating that conophylline-mediated inhibition of fibrosis is not specific to collagen synthesis. Conophylline affected neither TGFß-induced nuclear translocation of SMAD family member 2/3 (SMAD2/3) nor phosphorylation of SMAD2. However, conophylline substantially inhibited phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), suggesting that conophylline inhibits HAS2 expression via TGFß-mediated activation of the ERK1/2 pathway. Taken together, our results indicate that conophylline may be a useful inhibitor of ECM formation in fibrosis.

Chemical synthesis of 4-azido-ß-galactosamine derivatives for inhibitors of N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase.

Chondroitin sulfate E (CS-E) plays a crucial role in diverse processes ranging from viral infection to neuroregeneration. Its regiospecific sulfation pattern, generated by N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST), is the main structural determinant of its biological activity. Inhibitors of GalNAc4S-6ST can serve as powerful tools for understanding physiological functions of CS-E and its potential therapeutic leads for human diseases. A family of new 4-acylamino-ß-GalNAc derivatives and 4-azido-ß-GalNAc derivatives were synthesized for their potential application as inhibitors of GalNAc4S-6ST. The target compounds were evaluated for their inhibitory activities against GalNAc4S-6ST. The results revealed that 4-pivaloylamino- and 4-azido-ß-GalNAc derivatives displayed evident activities against GalNAc4S-6ST with IC50 value ranging from 0.800 to 0.828 mM. They showed higher activities than benzyl D-GalNAc4S that was used as control.

Versican A-subdomain is required for its adequate function in dermal development.

Versican, a large chondroitin sulfate (CS) proteoglycan, serves as a structural macromolecule of the extracellular matrix (ECM) and regulates cell behavior. We determined the function of versican in dermal development using VcanΔ3/Δ3 mutant mice expressing versican with deleted A-subdomain of the N-terminal G1 domain. The mutant versican showed a decreased hyaluronan (HA)-binding ability and failed to accumulate in the ECM. In the early developmental stage, VcanΔ3/Δ3 dermis showed a decrease in versican expression as compared with WT. As development proceeded, versican expression further decreased to a barely detectable level, and VcanΔ3/Δ3 mice died at the neonatal period (P0). At P0, VcanΔ3/Δ3 dermis exhibited an impaired ECM structure and decreased cell density. While the level of collagen deposition was similar in both genotypes, collagen biosynthesis significantly decreased in VcanΔ3/Δ3 fibroblasts as compared with that in wild type (WT). Transforming growth factor ß (TGFß) signaling mediated through the Smad2/3-dependent pathway was down-regulated in VcanΔ3/Δ3 fibroblasts and a reduced TGFß storage in the ECM was observed. Microarray analysis revealed a decrease in the expression levels of transcription factors, early growth response (Egr) 2 and 4, which act downstream of TGFß signaling. Thus, our results suggest that A-subdomain is necessary for adequate versican expression in dermis and that versican is involved in the formation of the ECM and regulation of TGFß signaling.

Sequence determination of synthesized chondroitin sulfate dodecasaccharides.

Chondroitin sulfate (CS) is a linear acidic polysaccharide composed of repeating disaccharide units of glucuronic acid and N-acetyl-d-galactosamine. The polysaccharide is modified with sulfate groups at different positions by a variety of sulfotransferases. CS chains exhibit various biological and pathological functions by interacting with cytokines and growth factors and regulating their signal transduction. The fine structure of the CS chain defines its specific biological roles. However, structural analysis of CS has been restricted to disaccharide analysis, hampering the understanding of the structure-function relationship of CS chains. Here, we chemo-enzymatically synthesized CS dodecasaccharides having various sulfate modifications using a bioreactor system of bacterial chondroitin polymerase mutants and various CS sulfotransferases. We developed a sequencing method for CS chains using the CS dodecasaccharides. The method consists of (i) labeling a reducing end with 2-aminopyridine (PA), (ii) partial digestion of CS with testicular hyaluronidase, followed by separation of PA-conjugated oligosaccharides with different chain lengths, (iii) limited digestion of these oligosaccharides with chondroitin lyase AC II into disaccharides, followed by labeling with 2-aminobenzamide, (iv) CS disaccharide analysis using a dual-fluorescence HPLC system (reversed-phase ion-pair and ion-exchange chromatography), and (v) estimation of the composition by calculating individual disaccharide ratios. This CS chain sequencing allows characterization of CS-modifying enzymes and provides a useful tool toward understanding the structure-function relationship of CS chains.

Host stromal versican is essential for cancer-associated fibroblast function to inhibit cancer growth.

The stroma provides a microenvironment that regulates tumor cell behavior. The extracellular matrix (ECM) has long been recognized to be important in tumor cell behavior, and previous studies have revealed the impact of individual matrix molecules on tumor progression. Although several reports have highlighted some central roles of tumor cell-expressed versican, the role of host stromal versican is not yet understood. In this study, we demonstrate that versican is an important molecule in the functional ECM structure and maintaining cancer-associated fibroblasts, using versican-negative QRsP11 fibrosarcoma cells. By their subcutaneous injection with cre-expressing adenoviruses to versican-floxed mice, we demonstrate that loss of host stromal versican facilitates tumor cell proliferation, and following angiogenesis, decreases cancer-associated fibroblasts, diminishes collagen fibers and alters hyaluronan distribution, concomitant with upregulation of hyaluronan, TGFß and VEGF-mediated signaling. When the versican V3 variant consisting of G1 and G3 domains was expressed in tumor cells, it was integrated into the ECM, regained collagen fibers and cancer-associated fibroblasts and resulted in successful recovery of tumor growth inhibition, indicating that whatever cells produce, the G1 and G3 domains are adequate for versican function. Collectively, our results indicate a dynamic function of versican in the ECM that regulates tumor cell behavior. A greater understanding of the regulation of versican expression may contribute to the development of cancer therapies.

Molecular dissection of placental malaria protein VAR2CSA interaction with a chemo-enzymatically synthesized chondroitin sulfate library.

Placental malaria, a serious infection caused by the parasite Plasmodium falciparum, is characterized by the selective accumulation of infected erythrocytes (IEs) in the placentas of the pregnant women. Placental adherence is mediated by the malarial VAR2CSA protein, which interacts with chondroitin sulfate (CS) proteoglycans present in the placental tissue. CS is a linear acidic polysaccharide composed of repeating disaccharide units of D-glucuronic acid and N-acetyl-D-galactosamine that are modified by sulfate groups at different positions. Previous reports have shown that placental-adhering IEs were associated with an unusually low sulfated form of chondroitin sulfate A (CSA) and that a partially sulfated dodecasaccharide is the minimal motif for the interaction. However, the fine molecular structure of this CS chain remains unclear. In this study, we have characterized the CS chain that interacts with a recombinant minimal CS-binding region of VAR2CSA (rVAR2) using a CS library of various defined lengths and sulfate compositions. The CS library was chemo-enzymatically synthesized with bacterial chondroitin polymerase and recombinant CS sulfotransferases. We found that C-4 sulfation of the N-acetyl-D-galactosamine residue is critical for supporting rVAR2 binding, whereas no other sulfate modifications showed effects. Interaction of rVAR2 with CS is highly correlated with the degree of C-4 sulfation and CS chain length. We confirmed that the minimum structure binding to rVAR2 is a tri-sulfated CSA dodecasaccharide, and found that a highly sulfated CSA eicosasaccharide is a more potent inhibitor of rVAR2 binding than the dodecasaccharides. These results suggest that CSA derivatives may potentially serve as targets in therapeutic strategies against placental malaria.

Sulfation of the bikunin chondroitin sulfate chain determines heavy chain·hyaluronan complex formation.

Inter-α-trypsin inhibitor (IαI) is a complex comprising two heavy chains (HCs) that are covalently bound by an ester bond to chondroitin sulfate (CS), which itself is attached to Ser-10 of bikunin. IαI is essential for the trans-esterification of HCs onto hyaluronan (HA). This process is important for the stabilization of HA-rich matrices during ovulation and some inflammatory processes. Bikunin has been isolated previously by anion exchange chromatography with a salt gradient up to 0.5 M NaCl and found to contain unsulfated and 4-sulfated CS disaccharides. In this study, bikunin-containing fractions in plasma and urine were separated by anion exchange chromatography with a salt gradient of 0.1-1.0 M NaCl, and fractions were analyzed for their reactivity with the 4-sulfated CS linkage region antibody (2B6). The fractions that reacted with the 2B6 antibody (0.5-0.8 M NaCl) were found to predominantly contain sulfated CS disaccharides, including disulfated disaccharides, whereas the fractions that did not react with this antibody (0.1-0.5 M NaCl) contained unsulfated and 4-sulfated CS disaccharides. IαI in the 0.5-0.8 M NaCl plasma fraction was able to promote the trans-esterification of HCs to HA in the presence of TSG-6, whereas the 0.1-0.5 M NaCl fraction had a much reduced ability to transfer HC proteins to HA, suggesting that the CS containing 4-sulfated linkage region structures and disulfated disaccharides are involved in the HC transfer. Furthermore, these data highlight that the structure of the CS attached to bikunin is important for the transfer of HC onto HA and emphasize a specific role of CS chain sulfation.

Subpopulations of fibroblasts derived from human iPS cells.

Organ fibrosis causes collagen fiber overgrowth and impairs organ function. Cardiac fibrosis after myocardial infarction impairs cardiac function significantly, pulmonary fibrosis reduces gas exchange efficiency, and liver fibrosis disturbs the natural function of the liver. Its development is associated with the differentiation of fibroblasts into myofibroblasts and increased collagen synthesis. Fibrosis has organ specificity, defined by the heterogeneity of fibroblasts. Although this heterogeneity is established during embryonic development, it has not been defined yet. Fibroblastic differentiation of induced pluripotent stem cells (iPSCs) recapitulates the process by which fibroblasts acquire diversity. Here, we differentiated iPSCs into cardiac, hepatic, and dermal fibroblasts and analyzed their properties using single-cell RNA sequencing. We observed characteristic subpopulations with different ratios in each organ-type fibroblast group, which contained both resting and distinct ACTA2+ myofibroblasts. These findings provide crucial information on the ontogeny-based heterogeneity of fibroblasts, leading to the development of therapeutic strategies to control fibrosis.

Periapical bacterial disinfection is critical for dental pulp regenerative cell therapy in apical periodontitis in dogs.

BACKGROUND: Application of pulp regenerative cell therapy for mature teeth with periapical lesions is a critical clinical challenge. The bacterial infection in inaccessible location within the root canal system and in the periapical lesions could cause resistance and impediment, leading to limitations in successful therapy. Thus, the aim of this study was to examine the effect of residual bacteria on the outcome of pulp regeneration in mature teeth with apical periodontitis in dogs. METHODS: Periapical lesions were induced in 32 root canals of 4 dogs in two different models in severities, model A and model B. Model A (moderate infection): the canal exposed to the oral cavity for 2 weeks and then closed for 2 weeks. Model B (severe infection): the canal exposed to the oral cavity for 2 months and then closed for 5 months. All root canals were irrigated with 6% sodium hypochlorite, and 3% EDTA and further with 0.015% levofloxacin-containing nanobubbles, which was also used as an intracanal medicament. The aseptic conditions were examined by bacterial anaerobic culture and/or PCR analyses. The root canal treatment was repeated several times, and allogeneic dental pulp stem cells were transplanted into the root canals. The radiographic evaluation of periapical lesions was performed by cone-beam computed tomography before the first treatment, just after cell transplantation, and after 2 months and 6 months in both model A, model B, respectively. The animals were then sacrificed and the jaw blocks were harvested for histological and histobacteriological evaluations of pulp regeneration and periapical tissue healing. Furthermore, the DiI-labelled DPSCs were transplanted into the root canals after complete disinfection (n = 4) or without root canal treatment (n = 4) in the apical periodontitis model (model A) in one dog, and cell localization was compared 72 h after transplantation. RESULTS: In 8 out of 12 canals from model A, and 10 out of 15 canals from model B, pulp regeneration with good vascularization, innervation, and a significant reduction in the radiolucent area of the periapical lesions were observed. However, in the other 4 canals and 5 canals from model A and model B, respectively, no pulp tissue was regenerated, and inflammation in the periapical tissue, and external resorption or healed external resorption were detected. The presence of residual bacteria in the periapical tissues and severe inflammation were significantly associated with inhibition of regenerated pulp tissue in these 9 unsuccessful canals (P < 0.05, each) (OR = 0.075, each) analyzed by multiple logistic regression analysis. For cellular kinetics, transplanted cells remained in the disinfected root canals, while they were not detected in the infected root canals, suggesting their migration through the apical foramen under the influence of inflammation. CONCLUSIONS: A true pulp-dentin complex was regenerated in the root canal by the pulp regenerative therapy in mature teeth with apical lesions. The successful pulp regeneration was negatively associated both with residual bacteria and inflammation in the periapical tissue.

Chondroitin sulfate-liposome: clustering toward high functional efficiency.

Chondroitin sulfate (CS) is a linear polysaccharide chain of alternating residues of glucuronic acid (GlcA) and N-acetylgalactosamine (GalNAc), modified with sulfate groups. Based on the structure, CS chains bind to bioactive molecules specifically and regulate their functions. For example, CS whose GalNAc is sulfated at the C4 position, termed CSA, and CS whose GalNAc is sulfated at both C4 and C6 positions, termed CSE, bind to a malaria protein VAR2CSA and receptor type of protein tyrosine phosphatase sigma (RPTPσ), respectively in a specific manner. Here, we modified CSA and CSE chains with phosphatidylethanolamine (PE) at a reducing end, attached them to liposomes containing phospholipids, and generated CSA- and CSE-liposomes. The CS-PE was incorporated into the liposome particles efficiently. Inhibition ELISA revealed specific interaction of CSA and CSE with recombinant VAR2CSA and RPTPσ, respectively, more efficiently than CS chains alone. Furthermore, CSE-liposome was specifically incorporated into RPTPσ-expressing HEK293T cells. These results indicate CS-liposome as a novel and efficient drug delivery system, especially for CS-binding molecules.

Construction of a chondroitin sulfate library with defined structures and analysis of molecular interactions.

Chondroitin sulfate (CS) is a linear acidic polysaccharide, composed of repeating disaccharide units of glucuronic acid and N-acetyl-D-galactosamine and modified with sulfate residues at different positions, which plays various roles in development and disease. Here, we chemo-enzymatically synthesized various CS species with defined lengths and defined sulfate compositions, from chondroitin hexasaccharide conjugated with hexamethylenediamine at the reducing ends, using bacterial chondroitin polymerase and recombinant CS sulfotransferases, including chondroitin-4-sulfotransferase 1 (C4ST-1), chondroitin-6-sulfotransferase 1 (C6ST-1), N-acetylgalactosamine 4-sulfate 6-sulfotransferase (GalNAc4S-6ST), and uronosyl 2-sulfotransferase (UA2ST). Sequential modifications of CS with a series of CS sulfotransferases revealed their distinct features, including their substrate specificities. Reactions with chondroitin polymerase generated non-sulfated chondroitin, and those with C4ST-1 and C6ST-1 generated uniformly sulfated CS containing >95% 4S and 6S units, respectively. GalNAc4S-6ST and UA2ST generated highly sulfated CS possessing ∼90% corresponding disulfated disaccharide units. Sequential reactions with UA2ST and GalNAc4S-6ST generated further highly sulfated CS containing a mixed structure of disulfated units. Surprisingly, sequential reactions with GalNAc4S-6ST and UA2ST generated a novel CS molecule containing ∼29% trisulfated disaccharide units. Enzyme-linked immunosorbent assay and surface plasmon resonance analysis using the CS library and natural CS products modified with biotin at the reducing ends, revealed details of the interactions of CS species with anti-CS antibodies, and with CS-binding molecules such as midkine and pleiotrophin. Chemo-enzymatic synthesis enables the generation of CS chains of the desired lengths, compositions, and distinct structures, and the resulting library will be a useful tool for studies of CS functions.

Chondroitinase from baculovirus Bombyx mori nucleopolyhedrovirus and chondroitin sulfate from silkworm Bombyx mori.

Chondroitin sulfate (CS) is a linear polysaccharide composed of repeating disaccharide units of glucuronic acid (GlcUA) and N-acetyl-d-galactosamine (GalNAc) with sulfate groups at various positions. Baculovirus is an insect-pathogenic virus that infects Lepidoptera larvae. Recently, we found that the occlusion-derived virus envelope protein 66 (ODV-E66) from Autographa californica nucleopolyhedrovirus (AcMNPV) exhibits chondroitin (CH)-digesting activity with distinct substrate specificity. Here, we demonstrate that the ODV-E66 protein from Bombyx mori nucleopolyhedrovirus (BmNPV) exhibits 92% homology to the amino acid sequence and 83% of the CH lyase activity of ODV-E66 from AcMNPV. ODV-E66 cleaves glycosyl bonds at nonreducing sides of disaccharide units consisting of nonsulfated and 6-O-sulfated GalNAc residues. We then investigated CS in the silkworm, Bombyx mori, which is the host of BmNPV. CS was present in insect tissues such as the midgut, peritrophic membrane, silk gland and skin. The polysaccharide consisted of a nonsulfated disaccharide unit, mono-sulfated disaccharide at Position 4 of the GalNAc residue and mono-sulfated disaccharide at Position 6 of the GalNAc residue. With regard to immunohistochemical analysis, the staining patterns of the silkworm tissues were different among anti-CS antibodies. Chondroitn sulfate that is digestible by ODV-E66 exists sufficiently in the peritrophic membrane protecting the midgut epithelium from ingested pathogens. Our results suggest that ODV-E66 facilitates the primary infection of the virus by digestion of CS in the peritrophic membrane.

Involvement of heparan sulfate 6-O-sulfation in the regulation of energy metabolism and the alteration of thyroid hormone levels in male mice.

Here, we report that male heparan sulfate 6-O-sulfotransferase-2 (Hs6st2) knockout mice showed increased body weight in an age-dependent manner even when fed with a normal diet and showed a phenotype of impaired glucose metabolism and insulin resistance. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that the expression of mitochondrial uncoupling proteins Ucp1 and Ucp3 was reduced in the interscapular brown adipose tissue (BAT) of male Hs6st2 knockout mice, suggesting reduced energy metabolism. The serum level of thyroid-stimulating hormone was significantly higher and that of thyroxine was lower in the knockout mice. When cultures of brown adipocytes from wild-type and Hs6st2 knockout mice isolated and differentiated in vitro were treated with FGF19 (fibroblast growth factor 19) or FGF21 in the presence or the absence of heparitinase I, phosphorylation of p42/p44 mitogen-activated protein (MAP) kinase was reduced. Heparan sulfate (HS) 6-O-sulfation was reduced not only in BAT but also in the thyroid tissue of the knockout mice. Thus, 6-O-sulfation in HS seems to play an important role in mediating energy metabolism by controlling thyroid hormone levels and signals from the FGF19 subfamily proteins, and the alteration of the HS composition may result in metabolic syndrome phenotypes such as altered glucose and insulin tolerance.

Versican provides the provisional matrix for uterine spiral artery dilation and fetal growth.

The extracellular matrix (ECM) in the endometrium plays a crucial role in mammalian pregnancy. We have shown that versican secreted from the endometrial epithelium promotes embryo implantation. Versican is a proteoglycan, a major player in the provisional matrix, and versikine, its N-terminal fragment cleaved by ADAMTS proteinases, serves as a bioactive molecule. Here, since versican expression in the placenta was dynamically altered in humans and mice, we investigated the role of versican in pregnancy using uterine-specific Vcan deletion mice (uKO mice) and ADAMTS-resistant versican expressing mice (V1R mice). uKO mice exhibited insufficient spiral artery dilation, followed by fetal growth restriction and maternal hypertension. Further analysis revealed impaired proliferation of tissue-resident natural killer cells required for spiral artery dilation. V1R mice showed the same results as the control, eliminating the involvement of versikine. Our results provide a new concept that versican, one factor of ECM, contributes to placentation and following fetal growth.

Keratan sulfate restricts neural plasticity after spinal cord injury.

Chondroitin sulfate (CS) proteoglycans are strong inhibitors of structural rearrangement after injuries of the adult CNS. In addition to CS chains, keratan sulfate (KS) chains are also covalently attached to some proteoglycans. CS and KS sometimes share the same core protein, but exist as independent sugar chains. However, the biological significance of KS remains elusive. Here, we addressed the question of whether KS is involved in plasticity after spinal cord injury. Keratanase II (K-II) specifically degraded KS, i.e., not CS, in vivo. This enzyme digestion promoted the recovery of motor and sensory function after spinal cord injury in rats. Consistent with this, axonal regeneration/sprouting was enhanced in K-II-treated rats. K-II and the CS-degrading enzyme chondroitinase ABC exerted comparable effects in vivo and in vitro. However, these two enzymes worked neither additively nor synergistically. These data and further in vitro studies involving artificial proteoglycans (KS/CS-albumin) and heat-denatured or reduced/alkylated proteoglycans suggested that all three components of the proteoglycan moiety, i.e., the core protein, CS chains, and KS chains, were required for the inhibitory activity of proteoglycans. We conclude that KS is essential for, and has an impact comparable to that of CS on, postinjury plasticity. Our study also established that KS and CS are independent requirements for the proteoglycan-mediated inhibition of axonal regeneration/sprouting.