Hyaluronan Binding Protein Research Articles

The extracellular matrix composition of the optic nerve subarachnoid space

The meninges not only surround the brain and the spinal cord but also the optic nerve. Meningeal-derived extracellular matrix (ECM) is a crucial component of the pial basement membrane, glia limitans and important for maintenance of optic nerve axon integrity, homeostasis and retinal ganglion cell health. To get closer insight into optic nerve meningeal-derived ECM composition, we performed proteomic analysis of the sheep optic nerve subarachnoid space (SAS). Candidate components were confirmed in cultures of primary human meningothelial cells (phMECs) and human optic nerve samples. Sheep optic nerve SAS samples were analysed by LC-MS, identified proteins were matched to their human orthologs and filtered using gene lists representing all major ECM components. To validate these findings digital droplet PCR (ddPCR) to evaluate mRNA expression of all candidate components identified was performed on cultures of phMECs. In addition, one protein per major ECM group was stained on human optic nerve sections and on phMEC cultures. Employing LC-MS, 1273 proteins were identified and subjected to bioinformatic analysis. Gene ontology analysis revealed six out of forty-four collagen types (1A1, 1A2, 3A1, 6A2, 6A3 and 14A1), three out of eleven laminin subunits (A4, B2, C1) and six out of twenty-seven hyaluronan binding proteins (CD44, versican (VCAN), C1q binding protein, neurocan (NCAN), brevican (BCAN) and hyalaluronan proteoglycan link protein 2 (HAPLN2)) were present in our cohort. DdPCR in phMEC cell culture confirmed presence of all candidate components except NCAN, BCAN and HAPLN2. Immunohistochemistry (IHC) staining on human optic nerve sections and immunofluorescence (IF) staining on in vitro cultured phMECs showed strong immunopositivity for collagen-typeI-α1 (COL1A1), lamininγ1 (LAMC1), and VCAN. Fibronectin (FN1) was exclusively present in cultures of phMECs. Using a combined bioinformatics and immunohistological approach, we describe the ECM composition of the optic nerve subarachnoid space. As this space plays an important role in maintaining optic nerve function, a better understanding of ECM composition in this delicate environment might be key to further pathophysiological insight into optic nerve degeneration and associated disorders.

Essential hyaluronan structure for binding with hyaluronan-binding protein (HABP) determined by glycotechnological approach

Hyaluronan specifically binds to aggrecan globular domain 1, which is often referred to as just hyaluronan binding protein (HABP), however, the hyaluronan carbohydrate structure recognized by HABP had not been studied in detail. The aim of the present study was to investigate the important structure of hyaluronan for binding to HABP. We prepared hybrid oligosaccharides from hyaluronan and chondroitin, with or without modification of the reducing or non-reducing terminus, as tools to determine the preferred structure of hyaluronan for binding to the HABP by a competitive ELISA-like method. The non-reducing terminal structure was critical, especially, the glucuronic acid (GlcUA) and N-acetylglucosamine (GlcNAc) of the hyaluronan-unit are essential for complete HABP binding activity, and for any HABP binding activity, respectively. It is possible to replace GlcUAβ-1-3GlcNAc of the internal disaccharide units with GlcUAβ-1-3N-acetylgalactosamine (GalNAc), if the chain length is decasaccharide or larger.

Detection of TSG-6-like protein in human corneal epithelium. Simultaneous presence with CD44 and hyaluronic acid

Tumor necrosis factor-inducible gene 6 protein (TSG-6) is member of the hyaluronan-binding protein family (hyaladherins) to which CD44 also belongs. Inflammatory mediators such as tumor necrosis factor α (TNF-α) and interleukin-1 (IL-1) stimulate TSG-6 production. Recently, however, externally applied TSG-6 has been shown to be effective in the treatment of inflammatory dry eye. On the other hand, it is still unknown whether TSG-6 is naturally present in human corneal epithelium. Corneal sections of 15 eyes enucleated for posterior segment uveal melanoma were immunohistochemically stained for hyaluronic acid (HA), CD44, and TSG-6. Throughout the corneal epithelium of all sections, CD44 and hyaluronic acid were detected most intensely in the basal epithelial layer. Whereas the presence of HA was intense even in the cytoplasm of the cells, CD44 was located predominantly at the cell membranes. The intensity of the specific staining decreased towards the surface, where CD44 was barely detectable. Hyaluronic acid was, on the other hand, detectable in the extracellular matrix and cells, even at the surface. TSG-6 like immunoreactivity was detected in all sections in a pattern similar to CD44 but much more distinct and intense, with a marked localization in the cell membranes and intercellular spaces, i.e., extracellular matrix. TSG-6 like immunoreactivity was clearly detectable through all cell layers of the corneal epithelium. All control sections were negative. Tumor necrosis factor-inducible gene 6 (TSG-6)- like protein is present in human corneal epithelium. It might be a natural component of this tissue which is constantly exposed and mechanically traumatized, and displays localization with similarities to that of CD44. The immunohistological detection of HA as major component of the ECM and epithelial tissue only confirms the results of earlier studies. However, the simultaneous presence and colocalization of CD44 and TSG-6, both HA-binding proteins, requires further investigation of the individual role, regulation and interaction of this system. The detection of TSG-6 in human corneal epithelium in the absence of inflammation underlines the importance of normal mechanical forces on the gene expression and regulation of this protein in ocular surface tissues. Given the relationship between inflammation and the protein, TSG-6 may be a major unknown and underestimated player in the regulation of the inflammation encountered in the presence of ocular surface desiccation and dry eye disease.

DEFB4A is a potential prognostic biomarker for colorectal cancer.

Colorectal cancer (CRC) is the third leading cause of cancer-associated mortality. The present study aimed to investigate novel biomarkers to predict prognosis and provide a theoretical basis for studies of the pathogenesis and the development of therapies for CRC. The present study compared mRNA expression levels of patients with CRC with short- and long-term prognosis and of individuals with and without tumors in The Cancer Genome Atlas (TCGA) database. Differentially expressed genes (DEGs) were identified via volcano plot and Venn diagram analysis. Gene Ontology (GO) analysis and gene set enrichment analysis (GSEA) were performed to identify the functions of the DEGs, and the DEGs were further verified using clinical CRC samples. A total of 10 DEGs were identified as candidate genes using the TCGA database, and four DEGs [defensin β 4A (DEFB4A), hyaluronan binding protein 2 (HABP2), oleoyl-ACP hydrolase and TBC1 domain family member 3G] were associated with poor prognosis of patients with CRC. Two DEGs (DEFB4A and HABP2) were upregulated in tumor tissues of patients with CRC in the TCGA database. GO and GSEA analyses revealed that DEFB4A was highly associated with immunosuppression, participates in ‘myeloid leukocyte differentiation’, ‘leukocyte proliferation’ and ‘positive regulation of leukocyte-mediated immunity’, and was positively correlated with CD11b, CD14, CD45, CD163 and IL17A. Furthermore, DEFB4A expression was significantly upregulated in patients with large tumors, advanced cancer stage, lymph node metastasis and liver metastasis. Survival analysis revealed that DEFB4A upregulation was associated with poor prognosis. DEFB4A gene knockdown experiments demonstrated that DEF4BA promotes cell migration. These results indicated that DEFB4A potentially promotes tumor growth by regulating immunosuppressive activity and provided novel insights into the diagnosis and treatment of CRC.

Involvement of hyaluronan in the adaptive changes of the rat small intestine neuromuscular function after ischemia/reperfusion injury

Intestinal ischemia/reperfusion (I/R) injury has severe consequences on myenteric neurons, which can be irreversibly compromised resulting in slowing of transit and hindered food digestion. Myenteric neurons synthesize hyaluronan (HA) to form a well-structured perineuronal net, which undergoes derangement when myenteric ganglia homeostasis is perturbed, i.e. during inflammation. In this study we evaluated HA involvement in rat small intestine myenteric plexus after in vivo I/R injury induced by clamping a branch of the superior mesenteric artery for 60 min, followed by 24 h of reperfusion. In some experiments, 4-methylumbelliferone (4-MU, 25 mg/kg), a HA synthesis inhibitor, was intraperitoneally administered to normal (CTR), sham-operated (SH) and I/R animals for 24 h. In longitudinal muscle myenteric plexus (LMMP) whole-mount preparations, HA binding protein staining as well as HA levels were significantly higher in the I/R group, and were reduced after 4-MU treatment. HA synthase 1 and 2 (HAS1 and HAS2) labelled myenteric neurons and mRNA levels in LMMPs increased in the I/R group with respect to CTR, and were reduced by 4-MU. The efficiency of the gastrointestinal transit was significantly reduced in I/R and 4-MU-treated I/R groups with respect to CTR and SH groups. In the 4-MU-treated I/R group gastric emptying was reduced with respect to the CTR, SH and I/R groups. Carbachol (CCh) and electrical field (EFS, 0.1–40 Hz) stimulated contractions and EFS-induced (10 Hz) NANC relaxations were reduced in the I/R group with respect to both CTR and SH groups. After I/R, 4-MU treatment increased EFS contractions towards control values, but did not affect CCh-induced contractions. NANC on-relaxations after I/R were not influenced by 4-MU treatment. Main alterations in the neurochemical coding of both excitatory (tachykinergic) and inhibitory pathways (iNOS, VIPergic) were also observed after I/R, and were influenced by 4-MU administration. Overall, our data suggest that, after an intestinal I/R damage, changes of HA homeostasis in specific myenteric neuron populations may influence the efficiency of the gastrointestinal transit. We cannot exclude that modulation of HA synthesis in these conditions may ameliorate derangement of the enteric motor function preventing, at least in part, the development of dysmotility.

Predicting Gemcitabine Delivery by 18F-FAC PET in Murine Models of Pancreatic Cancer.

18F-FAC (2'-deoxy-2'-18F-fluoro-β-d-arabinofuranosylcytosine) has close structural similarity to gemcitabine and thus offers the potential to image drug delivery to tumors. We compared tumor 18F-FAC PET images with 14C-gemcitabine levels, established ex vivo, in 3 mouse models of pancreatic cancer. We further modified tumor gemcitabine levels with injectable PEGylated recombinant human hyaluronidase (PEGPH20) to test whether changes in gemcitabine would be tracked by 18F-FAC. Methods:18F-FAC was synthesized as described previously. Three patient-derived xenograft (PDX) models were grown in the flanks of NSG mice. Mice were given PEGPH20 or vehicle intravenously 24 h before coinjection of 18F-FAC and 14C-gemcitabine. Animals were euthanized and imaged 1 h after tracer administration. Tumor and muscle uptake of both 18F-FAC and 14C-gemcitabine was obtained ex vivo. The efficacy of PEPGPH20 was validated through staining with hyaluronic acid binding protein. Additionally, an organoid culture, initiated from a KPC (Pdx-1 Cre LSL-KrasG12D LSL-p53R172H) tumor, was used to generate orthotopically growing tumors in C57BL/6J mice, and these tumors were then serially transplanted. Animals were injected with PEGPH20 and 14C-gemcitabine as described above to validate increased drug uptake by ex vivo assay. PET/MR images were obtained using a PET insert on a 7-T MR scanner. Animals were imaged immediately before injection with PEGPH20 and again 24 h later. Results: Tumor-to-muscle ratios of 14C-gemcitabine and 18F-FAC correlated well across all PDX models and treatments (R2 = 0.78). There was a significant increase in the tumor PET signal in PEGPH20-treated PDX animals, and this signal was matched in ex vivo counts for 2 of 3 models. In KPC-derived tumors, PEGPH20 raised 14C-gemcitabine levels (tumor-to-muscle ratio of 1.9 vs. 2.4, control vs. treated, P = 0.013). PET/MR 18F-FAC images showed a 12% increase in tumor 18F-FAC uptake after PEGPH20 treatment (P = 0.023). PEGPH20-treated animals uniformly displayed clear reductions in hyaluronic acid staining. Conclusion:18F-FAC PET was shown to be a good surrogate for gemcitabine uptake and, when combined with MR, to successfully determine drug uptake in tumors growing in the pancreas. PEGPH20 had moderate effects on tumor uptake of gemcitabine.

Combining RNA in situ hybridization and spectral flow cytometry to investigate the leukocyte glycocalyx in autoimmunity

Abstract The glycocalyx surrounding leukocytes has been studied for its role in immunomodulation and its potential as a therapeutic target. 4-methylumbelliferone (4MU) is a small molecule that inhibits production of the glycocalyx’s primary polysaccharide component hyaluronan (HA), by targeting the HA synthase (HAS). Oral treatment with 4MU has proved efficacious in preventing the development and progression of autoimmunity, though the mechanism is contested. We have previously shown that 4MU strongly inhibits T-cell mediated autoimmunity, and report here that it also attenuates formation of antigen-specific plasmablasts and subsequent autoantibody production. This suggests that the HA glycocalyx plays a role in promoting the humoral component of autoimmunity. The HAS proteins are deeply membrane embedded, preventing the use of traditional flow cytometry antibodies to define expression levels across leukocyte compartments. This limitation has made it difficult to identify cellular targets of 4MU. Alternative measurements must be used to study the HA glycocalyx. Surface HA content of a cell can be measured with labelled HA binding protein (HABP), though this does not explain whether HA was synthesized or scavenged with HA-binding receptors. RNA measurement can also be used to look at transcript levels of the HAS proteins. Here, we combine RNA in situ hybridization, fluorescently labeled HA binding protein (HABP), and conventional flow cytometry antibodies to define the HA glycocalyx on leukocytes during autoimmunity. Utilizing Spectral Flow Cytometry, we can assess parameters in numbers comparable to Mass Cytometry (CyTOF) to identify which cells are most likely targeted by 4MU to prevent antibody formation during autoimmunity.

Mycosporine-like amino acids stimulate hyaluronan secretion by up-regulating hyaluronan synthase 2 via activation of the p38/MSK1/CREB/c-Fos/AP-1 axis

Hyaluronan (HA) is an extracellular matrix glycosaminoglycan that critically supports the physicochemical and mechanical properties of the skin. Here, we demonstrate that mycosporine-like amino acids (MAAs), which typically function as UV-absorbing compounds, can stimulate HA secretion from normal human fibroblasts. MAA-stimulated HA secretion was associated with significantly increased and decreased levels of mRNAs encoding HA synthase 2 (HAS2) and the HA-binding protein involved in HA depolymerization (designated HYBID), respectively. Using immunoblotting, we found that MAAs at 10 and at 25 μg/ml stimulate the phosphorylation of the mitogen-activated protein kinase (MAPK) p38, extracellular signal-regulated kinase (ERK)/c-Jun, and mitogen- and stress-activated protein kinase 1 (MSK1) (at Thr-581, Ser-360, and Ser-376, respectively) and activation of cAMP-responsive element-binding protein (CREB) and activating transcription factor 2 (ATF2), but not phosphorylation of JUN N-terminal kinase (JNK) or NF-κB (at Ser-276 or Ser-536, respectively), and increased c-Fos protein levels. Moreover, a p38-specific inhibitor, but not inhibitors of MAPK/ERK kinase (MEK), JNK, or NF-κB, significantly abrogated the increased expression of HAS2 mRNA, accompanied by significantly decreased MAA-stimulated HA secretion. These results suggested that the p38-MSK1-CREB-c-Fos-transcription factor AP-1 (AP-1) or the p38-ATF2 signaling cascade is responsible for the MAA-induced stimulation of HAS2 gene expression. Of note, siRNA-mediated ATF2 silencing failed to abrogate MAA-stimulated HAS2 expression, and c-Fos silencing abolished the increased expression of HAS2 mRNA. Our findings suggest that MAAs stimulate HA secretion by up-regulating HAS2 mRNA levels through activation of an intracellular signaling cascade consisting of p38, MSK1, CREB, c-Fos, and AP-1.

RETRACTED: circ_001653 Silencing Promotes the Proliferation and ECM Synthesis of NPCs in IDD by Downregulating miR-486-3p-Mediated CEMIP

Functional changes of nucleus pulposus cells (NPCs) are considered to be the initiating factors of intervertebral disc degeneration (IDD). In this study, we investigated whether circular RNA homo sapiens (hsa)_circ_001653 (circ_001653) could bind to microRNA-486-3p (miR-486-3p) to regulate the biological properties of NPCs and the synthesis of extracellular matrix (ECM) in IDD. Initially, circ_001653 was highly expressed in isolated NPCs and degenerative NP tissues in close relation to the severity of IDD. To evaluate the effects of circ_001653 on cellular processes, we performed experiments in vitro and in vivo with altered expression of circ_001653 and miR-486-3p. An increased expression of circ_001653 in the NPCs and the degenerative NP tissues was directly associated with elevated apoptosis and an imbalance between anabolic and catabolic factors of the ECM. miR-486-3p regulated NPC proliferation and inhibited the expression of CEMIP, the cell migration-inducing hyaluronan binding protein. circ_001653 regulated miR-486-3p expression, functioning in NPCs to upregulate CEMIP, whereas circ_001653 silencing alleviated IDD in the mouse model. Altogether, circ_001653 downregulation could potentially alleviate NPC apoptosis and the metabolic imbalance of the ECM through the miR-486-3p/CEMIP axis. These mechanistic insights may present new therapeutic targets for the treatment of IDD.

TLR2/4 promotes PGE2 production to increase tissue damage in Escherichia coli-infected bovine endometrial explants via MyD88/p38 MAPK pathway

Prostaglandin E2 (PGE2), a lipid mediator, is released by several cell types including endometrial cells and plays a central role in bacterial infection of the endometrium during inflammation. PGE2 production accumulated in Escherichia coli (E. coli) -infected bovine endometrial tissue, which increased E. coli-infected endometrial tissue damage. However, the mechanisms of PGE2 accumulation in the E. coli-infected endometrium during inflammation-associated endometrial tissue damage remain unclear. This study was conducted to investigate the role of Toll-like receptors (TLRs) 2 and 4 in increased PGE2 production in E. coli-infected endometrial tissue. E. coli and TLR2/4 agonists significantly induced cyclooxygenase-2 and microsomal prostaglandin E synthase-1 expression and PGE2 synthesis detected by RT-PCR, Western blot, and ELISA in the endometrial tissue. The expression and synthesis were dramatically decreased by TLR4, myeloid differentiation factor88 (MyD88), and p38 mitogen-activated protein kinase (MAPK) inhibitors in E. coli-infected endometrial tissue. These inhibitors also significantly decreased proinflammatory factor (interleukin-6 and tumor necrosis factor-α) and damage-associated molecular pattern (high mobility group box-1 and hyaluronan-binding protein-1) release and tissue damage measured by double-label immunofluorescence in E. coli-infected endometrial explants. Our work provides in vitro evidence that TLR2/4-MyD88/p38 MAPK promotes PGE2 synthesis and E. coli-infected endometrial tissue damage, which may be useful for improving PGE2-based therapies for endometritis.

Relative Shedding of Glycosaminoglycans from the Endothelial Glycocalyx During Inflammation

The endothelial (EC) surface layer (glycocalyx) has been shown to act as a barrier to transvascular exchange of solutes, and adhesion of leukocytes (WBCs) during the inflammatory process. It is a labile structure whose components are readily shed by the action of proteases and endoglycosidases. Evidence to date suggests that during the inflammatory process, activation of matrix metalloproteases on the EC surface may cleave the principal proteoglycans (syndecans and glypicans), and EC derived lyases may result in shedding of the associated glycosaminoglycans (GAGs) heparan sulfate (HS), chondroitin sulfate (CS) and hyaluronan (HA). Shedding of these components of the glycocalyx serves to increase leukocyte (WBC) access to adhesion receptors on the EC membrane. The objective of the present study was to elucidate the relative role of HS (the dominant GAG) and HA as the major barrier to WBC‐EC adhesion.Shedding of glycans from the EC in post‐capillary venules of the mesentery (rat) in response to topical application of the chemoattractant f‐met‐leu‐phe (fMLP, 10−6 M) was observed by intravital microscopy. The presence and loss of HS, CS and HA was assessed by labeling all components with fluorescently labelled lectin (BS‐1) or HS antibodies, and HA with fluorescently labelled hyaluronan binding protein (HBP). During exposure to fMLP, GAG concentration exhibited an exponential decay. Following a 30 min period about 50% of HBP was lost in contrast to a previously shown loss of 20% of lectin labelled GAGs, and 25% loss of Mab labelled HS. The exponential time‐constant for HBP shedding was 5.8 min, which was one‐third the 14.3 min for BS‐1 labelled GAGs.The relatively rapid shedding of HA compared to HS was consistent with the hypothesis that HA may form the dominant barrier to WBC‐EC adhesion. In situ observations (in rat mesentery) of fMLP induced transient adhesion of circulating beads coated with antibody to ICAM‐1 (1A29) revealed a time constant for adhesion of 10.9 min, which was less than that for BS‐1 labelled GAGs. Prior observations by Fan et al., (Biorheology, 2019, DOI: 10.3233/BIR‐180204) using stochastic optical reconstruction microscopy suggest that HA lies closer to and parallel to the endothelial membrane, compared to HS which lies further away and normal to the EC surface. Thus, a compact layer of HA near the EC membrane may surround WBC adhesion receptors that are much shorter in length than the total thickness of the glycocalyx. Its more rapid shedding, compared to other GAGs, may be the major factor affecting exposure of WBC adhesion receptors on the EC.Support or Funding InformationSupported in part by NIH R01 HL39286

MP31-11 HYALURONIC ACID DEGRADING ENZYME

INTRODUCTION AND OBJECTIVE: The etiology and pathophysiology of interstitial cystitis/bladder pain syndrome (IC/BPS) has not been elucidated. One cause of IC/BPS is considered to be the abnormal glycosaminoglycan layer that acts as a chemoprotective factor in the urothelium. Therefore, replacement therapy with mucopolysaccharides such as hyaluronic acid (HA) has been performed. In this study, we focused on HA which is a urothelial protective factor and investigated the metabolism of intravesical HA in IC/BPS patient. METHODS: The RNA expression levels of HA synthase and HA degrading enzyme were analyzed using qPCR from the urinary cell-derived RNA of 10 IC/BPS patients and 10 healthy subjects. We reviewed the following enzymes: HA synthase 1 (HAS1), HAS2 and HAS3. We also reviewed the following HA-degrading enzymes: hyaluronidase 1 (HYAL1), HYAL2, transmembrane protein 2 (TMEM2), cell migration-inducing and hyaluronan-binding protein (CEMIP). RESULTS: The levels of HA synthases such as HAS1, HAS2, and HAS3 in the IC/BPS patients were not significantly different from those in healthy subjects. The level of HA-degrading enzyme, TMEM2 gene, was significantly higher in IC/BPS patients (p <0.0001). The level of the other HA-degrading enzymes (HYAL1, HYAL2, CEMIP) were not significantly different between the two groups. CONCLUSIONS: These results suggest that the synthesis of HA in IC/BPS patients is not different from that in healthy individuals. However, the level of HA degradation enzyme (especially TMEM2) is greater in IC/BPS patients than in healthy individuals. Therefore, TMEM2 is a potential diagnostic and therapeutic marker for IC/BPS.Source of Funding: none

CEMIP regulates the proliferation and migration of vascular smooth muscle cells in atherosclerosis through the WNT-beta-catenin signaling pathway.

In this study we investigated the regulatory role of cell-migration-inducing and hyaluronan-binding protein (CEMIP) in the proliferation and migration of vascular smooth muscle cells (VSMCs). The mRNA and protein levels of CEMIP were upregulated in the plasma samples from patients with atherosclerosis, and in VSMCs stimulated with platelet-derived growth factor-BB (PDGF-BB), compared with plasma from healthy subjects and untreated VSMCs. Silencing CEMIP suppressed PDGF-BB-induced cell migration and proliferation in VSMCs, as determined using a Cell Counting Kit-8 assays, 5-ethynyl-2'-deocyuridine (EDU) assays, flow cytometry, wound healing assays, and Transwell assays. Overexpression of CEMIP promoted the proliferation and migration of VSMCs via activation of the Wnt-β-catenin signaling pathway and the upregulation of its target genes, including matrix metalloproteinase-2, matrix metalloproteinase-7, cyclin D1, and c-myc, whereas CEMIP deficiency showed the opposite effects. The knockdown of CEMIP in ApoE-/- mice by intravenous injection of lentiviral vector expressing si-CEMIP protected against high-fat-diet-induced atherosclerosis, as shown by the reduced aortic lesion areas, aortic sinus lesion areas, and the concentration of blood lipids compared with mice normally expressing CEMIP. These results demonstrated that CEMIP regulates the proliferation and migration of VSMCs in atherosclerosis by activating the WNT-β-catenin signaling pathway, which suggests the therapeutic potential of CEMIP for the management of atherosclerosis.

Sirtuin 1 reduces hyaluronan synthase 2 expression by inhibiting nuclear translocation of NF-κB and expression of the long-noncoding RNA HAS2–AS1

Hyaluronan (HA) is one of the most prevalent glycosaminoglycans of the vascular extracellular matrix (ECM). Abnormal HA accumulation within blood vessel walls is associated with tissue inflammation and is prominent in most vascular pathological conditions such as atherosclerosis and restenosis. Hyaluronan synthase 2 (HAS2) is the main hyaluronan synthase enzyme involved in HA synthesis and uses cytosolic UDP-glucuronic acid and UDP-GlcNAc as substrates. The synthesis of UDP-glucuronic acid can alter the NAD+/NADH ratio via the enzyme UDP-glucose dehydrogenase, which oxidizes the alcohol group at C6 to the COO− group. Here, we show that HAS2 expression can be modulated by sirtuin 1 (SIRT1), the master metabolic sensor of the cell, belonging to the class of NAD+-dependent deacetylases. Our results revealed the following. 1) Treatments of human aortic smooth muscle cells (AoSMCs) with SIRT1 activators (SRT1720 and resveratrol) inhibit both HAS2 expression and accumulation of pericellular HA coats. 2) Tumor necrosis factor α (TNFα) induced HA-mediated monocyte adhesion and AoSMC migration, whereas SIRT1 activation prevented immune cell recruitment and cell motility by reducing the expression levels of the receptor for HA-mediated motility, RHAMM, and the HA-binding protein TNF-stimulated gene 6 protein (TSG6). 3) SIRT1 activation prevented nuclear translocation of NF-κB (p65), which, in turn, reduced the levels of HAS2–AS1, a long-noncoding RNA that epigenetically controls HAS2 mRNA expression. In conclusion, we demonstrate that both HAS2 expression and HA accumulation by AoSMCs are down-regulated by the metabolic sensor SIRT1.

Effect of functional lateral shift of the mandible on hyaluronic acid metabolism related to lubrication of temporomandibular joint in growing rats.

Hyaluronic acid (HA) is a major molecular component of the articular cartilage of the temporomandibular joint (TMJ) influencing joint lubrication. Functional lateral shift of the mandible (FLSM) can lead to malocclusion. This study investigated the effects of FLSM on HA metabolism and lubrication of the TMJ in growing rats. Thirty 5-week-old male Wistar rats were divided into shift, recovery, and control groups. Rats in the shift and recovery groups were fitted with guiding plates to produce a 2-mm FLSM which were removed from the rats in the recovery group 14 days later. Animals were sacrificed at 14 and 28 days after the appliances were attached. Immunohistochemistry of HA-binding protein (HABP), hyaluronan synthase (HAS), and hyaluronoglucosaminidases (HYALs) was examined. The thickness of HABP-positively stained areas in the lateral regions in the bilateral condyle was reduced during the experimental period in the shift group compared with that in the control group. The proportion of HAS2-stained areas was bilaterally decreased in different regions of condylar cartilage during the experimental period in the shift group. The reduction of the HYAL2-stained area proportion in the condylar cartilage was more significant than that of HYAL1 at 14 days after appliance attachment in the shift group. HAS2 staining was not recovered in the recovery group. This research was based on animal experiments with a limited experimental period. FLSM altered lubrication related HA metabolism in the articular cartilage of the TMJ in growing rats.

Hyaluronan-binding protein 1 (HABP1) overexpression triggers induction of senescence in fibroblasts cells.

Hyaluronan-binding protein 1 (HABP1), a multi-compartmental, multi-functional protein has a wide range of functions, which can be attributed to its ability to associate with a variety of cellular ligands. Earlier we have reported that HABP1 overexpression in rat normal fibroblasts (F-HABP07) shows chronic generation of reactive oxygen species (ROS), induction of autophagy, and apoptosis. However, a significant proportion of cells remained viable after the majority went through apoptosis from 60 to 72 h. In this study, an attempt has been made to delineate the cellular events in the declined population of surviving cells. It has been elucidated here that, these cells at later time points of growth, that is, 72 and 84 h, not only appeared to shrink but also are devoid of autophagic vacuoles and displayed polyploidy. F-HABP07 cells exhibited an altered cytoskeletal structure from their parental cell line F111, assumed to be caused upon inhibition of actin polymerization and decrease in IQ motif-containing GTPase activating protein 1 (IQGAP1), a key protein associated with maintenance of cytoskeletal integrity. Enhanced expression and nuclear localization of AKT observed in F-HABP07 cells appears to be contributing toward the maintenance of high ROS levels in these cells and also potentially modulating the IQGAP1 activity. These observations, in fact have been considered to result in sustained DNA damage, which then leads to increased expression of p53 and activation of p21 and carry out the cellular events responsible for senescence. Subsequent assessment of the presence of positive β-gal staining and enhanced expression of p16INK4a in F-HABP07, confirmed that HABP1 overexpressing fibroblasts undergo senescence.

Implication of HYBID (Hyaluronan-Binding Protein Involved in Hyaluronan Depolymerization) in Hyaluronan Degradation by Synovial Fibroblasts in Patients with Knee Osteoarthritis

Cell migration-inducing hyaluronidase 1 (CEMIP), also known as hyaluronan (HA)-binding protein involved in HA depolymerization (HYBID), plays a role in HA degradation. CEMIP2, also known as transmembrane protein 2 (TMEM2), possessing a sequence similarity with HYBID, is reported as a hyaluronidase in mice. However, the expression of these molecules in osteoarthritic synovium and their involvement in HA degradation in synovial fluid (SF) from patients with knee osteoarthritis remain elusive. This study examined their expression in synovial tissue and the relationship with molecular weight of HA in SF in knee osteoarthritis patients. Quantification of mRNA demonstrated that HYBID expression is significantly (5.5-fold) higher in osteoarthritic synovium than in normal control synovium, whereas TMEM2 expression level is similar between the two groups. By immunohistochemistry, HYBID was localized mainly to CD68-negative and fibroblast-specific protein 1-positive synovial lining cells and sublining fibroblasts in osteoarthritic synovium. The mRNA expression levels of HYBID, but not TMEM2, in osteoarthritic synovium positively correlated with distribution of lower-molecular-weight HA with below 1000 kDa in SF. HA-degrading activity in osteoarthritic synovial fibroblasts was abrogated by siRNA-mediated knockdown of HYBID. Among the 12 factors examined, IL-6 significantly up-regulated the HYBID expression and HA-degrading activity in osteoarthritic synovial fibroblasts. These data suggest that HYBID overexpressed by IL-6-stimulated synovial fibroblasts is implicated in HA degradation in osteoarthritic synovium.

HYBID (alias KIAA1199/CEMIP) and hyaluronan synthase coordinately regulate hyaluronan metabolism in histamine-stimulated skin fibroblasts

The immune-regulatory compound histamine is involved in the metabolism of the essential skin component hyaluronan (HA). We previously reported that histamine up-regulates the expression of HYBID (hyaluronan-binding protein involved in hyaluronan depolymerization, also called CEMIP or KIAA1199), which plays a key role in HA degradation. However, no information is available about histamine's effects on HA synthase (HAS) expression, the molecular sizes of HA species produced, and histamine receptors and their signaling pathways in skin fibroblasts. Moreover, histamine's effects on photoaged skin remain elusive. Here, we show that histamine increases HA degradation by up-regulating HYBID and down-regulating HAS2 in human skin fibroblasts in a dose- and time-dependent manner and thereby decreases the total amounts and sizes of newly produced HA. Histamine H1 blocker abrogated the histamine effects on HYBID up-regulation, HAS2 suppression, and HA degradation. Histamine H1 agonist exhibited effects on HA levels, composition, and breakdown similar to those of histamine. Of note, blockade of protein kinase Cδ or PI3K-Akt signaling abolished histamine-mediated HYBID stimulation and HAS2 suppression, respectively. Immunohistochemical experiments revealed a significant ∼2-fold increase in tryptase-positive mast cells in photoaged skin, where HYBID and HAS2 expression levels were increased and decreased, respectively, compared with photoprotected skin. These results indicate that histamine controls HA metabolism by up-regulating HYBID and down-regulating HAS2 via distinct signaling pathways downstream of histamine receptor H1. They further suggest that histamine may contribute to photoaged skin damage by skewing HA metabolism toward degradation.

Overexpression of hyaluronan-binding protein 1 promotes the proliferation, migration, and invasion of malignant glioma.

BackgroundHyaluronan binding protein 1 (HABP1) is a member of the hyaladherin family and is involved in T cell activation. It is a high-grade polysaccharide. The CD44 family is also a member of this family. It is expressed in many tumor cells at higher levels than the corresponding normal tissues. It is related to the occurrence and development of tumor cells, invasiveness, and lymph node metastasis. However, the mechanism of HABP1 in glioma remains unclear.MethodsWe focused on the proliferation and invasion of HABP1 in gliomas. The expression of HABP1 was firstly investigated by immunohistochemical staining and by comparing different grades of glioma tissues from 80 patients with glioma. Then, the influence of migration and proliferation through different human glioma cell line models were investigated.ResultsBy collecting a large number of clinical pathological tissues and patient data, it was found that HABP1 expression was significantly correlated with glioma, and gels with a high expression of HABP1 and a low expression of HABP1 were screened by different glioma cell lines. In the cytomorphological analysis, short interfering RNA with HABP1 significantly inhibited the proliferation of glioma, and the overexpression of HABP1 enhanced glioma invasion and migration.ConclusionsHABP1 is likely to become a new targeting factor for the treatment of glioma. The mechanism of action of HABP1 in glioma remains to be further studied.

Expression and Characterization of Hyaluronan-Binding Protein Involved in Hyaluronan Depolymerization: HYBID, Alias KIAA1199 and CEMIP.

Hyaluronan (HA), a major component of the extracellular matrix in vertebrate tissues, provides structural and functional integrity to cells and organs. Biological functions of HA are dependent on the molecular size of HA and the interaction with a wide range of HA-binding proteins, i.e., hyaladherins. In this book chapter, we introduce hyaladherins and focus on HYBID (Hyaluronan-binding protein involved in hyaluronan depolymerization, alias KIAA1199 and CEMIP), which is one of the hyaladherins and plays a central role in HA degradation in human dermal and arthritic synovial fibroblasts. The protocols describe the preparation of the stable transfectants expressing HYBID, the assays of HYBID-mediated HA depolymerization, and the binding assay of HYBID to HA. These methods will be helpful to further study the HYBID-mediated biological activities and its relevance on HA degradation and turnover under various physiological and pathological conditions such as wound healing, ageing, arthritis, and cancer.