Hyaluronic acid synthesis, degradation, and crosslinking in equine osteoarthritis: TNF-α-TSG-6-mediated HC-HA formation

Background TNF-α-stimulated gene 6 (TSG-6) protein, a TNF-α-responsive hyaladherin, possesses enzymatic activity that can catalyze covalent crosslinks of the polysaccharide hyaluronic acid (HA) to another protein to form heavy chain-hyaluronic acid (HC-HA) complexes in pathological conditions such as osteoarthritis (OA). Here, we examined HA synthase and inflammatory gene expression; synovial fluid HA, TNF-α, and viscosity; and TSG-6-mediated HC-HA complex formation in an equine OA model. The objectives of this study were to (1) evaluate the TNF-α-TSG-6-HC-HA signaling pathway across multiple joint tissues, including synovial membrane, cartilage, and synovial fluid, and (2) determine the impact of OA on synovial fluid composition and biophysical properties. Methods HA and inflammatory cytokine concentrations (TNF-α, IL-1β, CCL2, 3, 5, and 11) were analyzed in synovial fluid from 63 OA and 25 control joints, and HA synthase (HAS1-3), TSG-6, and hyaluronan-degrading enzyme (HYAL2, HEXA) gene expression was measured in synovial membrane and cartilage. HA molecular weight (MW) distributions were determined using agarose gel electrophoresis and solid-state nanopore measurements, and HC-HA complex formation was detected via immunoblotting and immunofluorescence. SEC-MALS was used to evaluate TSG-6-mediated HA crosslinking, and synovial fluid and HA solution viscosities were analyzed using multiple particle-tracking microrheology and microfluidic measurements, respectively. Results TNF-α concentrations were greater in OA synovial fluid, and TSG6 expression was upregulated in OA synovial membrane and cartilage. TSG-6-mediated HC-HA complex formation was greater in OA synovial fluid and tissues than controls, and HC-HA was localized to both synovial membrane and superficial zone chondrocytes in OA joints. SEC-MALS demonstrated macromolecular aggregation of low MW HA in the presence of TSG-6 and inter-α-inhibitor with concurrent increases in viscosity. Conclusions Synovial fluid TNF-α concentrations, synovial membrane and cartilage TSG6 gene expression, and HC-HA complex formation were increased in equine OA. Despite the ability of TSG-6 to induce macromolecular aggregation of low MW HA with resultant increases in the viscosity of low MW HA solutions in vitro, HA concentration was the primary determinant of synovial fluid viscosity rather than HA MW or HC-HA crosslinking. The TNF-α-TSG-6-HC-HA pathway may represent a potential therapeutic target in OA.

CircHYBID regulates hyaluronan metabolism in chondrocytes via hsa-miR-29b-3p/TGF-β1 axis

Background Hyaluronan (HA) metabolism by chondrocytes is important for cartilage development and homeostasis. However, information about the function of circular RNAs (circRNAs) in HA metabolism is limited. We therefore profiled the role of the novel HA-related circRNA circHYBID in the progression of osteoarthritis (OA). Methods CircHYBID function in HA metabolism in chondrocytes was investigated using gain-of-function experiments, and circHYBID mechanism was confirmed via bioinformatics analysis and luciferase assays. The expression of circHYBID–hsa-miR-29b-3p–transforming growth factor (TGF)-β1 axis was examined by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. CircHYBID, TGF-β1, and HA levels in cartilage samples were evaluated using qRT-PCR and pathological examination. Enzyme-linked immunosorbent assay was used to assess HA accumulation in chondrocyte supernatant. Results CircHYBID expression was significantly downregulated in damaged cartilage samples compared with that in the corresponding intact cartilage samples. CircHYBID expression was positively correlated with alcian blue score. Interleukin-1β stimulation in chondrocytes downregulated circHYBID expression and decreased HA accumulation. Gain-of-function experiments revealed that circHYBID overexpression in chondrocytes increased HA accumulation by regulating HA synthase 2 and HYBID expression. Further mechanism analysis showed that circHYBID upregulated TGF-β1 expression by sponging hsa-miR-29b-3p. Conclusions Our results describe a novel HA-related circRNA that could promote HA synthesis and accumulation. The circHYBID–hsa-miR-29b-3p–TGF-β1 axis may play a powerful regulatory role in HA metabolism and OA progression. Thus, these findings will provide new perspectives for studies on OA pathogenesis, and circHYBID may serve as a potential target for OA therapy.

1-Ethyl-β-N-acetylglucosaminide increases hyaluronan production in human keratinocytes by being converted to N-acetylglucosamine via β-N-acetylglucosaminidase-dependent manner

Regulation of hyaluronan (HA) is important for the maintenance of epidermal homeostasis. Here we examined the mechanism by which 1-ethyl-β-N-acetylglucosaminide (β-NAG2), a newly developed N-acetylglucosamine (NAG) derivative, increases HA production in cultured human epidermal keratinocytes. When keratinocytes were treated with β-NAG2, mRNA expression of HA synthase 3, which is responsible for HA production in human keratinocytes, was not influenced, but the intracellular level of UDP-NAG, a substrate used for HA synthesis, was increased. By using a synthetic substrate for β-N-acetylglucosaminidase (β-NAGase), keratinocytes were found to possess β-NAGase activity, and treatment of o-(2-acetamido-2-deoxy-D-glucopyranosylidene) amino N-phenyl carbamate (PUGNAc), an inhibitor of β-NAGase, abolished the release of NAG from β-NAG2 in keratinocytes. Furthermore, PUGNAc attenuated the β-NAG2-induced intracellular UDP-NAG and HA production in keratinocytes. These results suggest that β-NAG2 is converted to NAG by endogenous β-NAGase in keratinocytes, and the resulting NAG is further metabolized to UDP-NAG and utilized for HA production.

C1q–HA Matrix Regulates the Local Synthesis of Hyaluronan in Malignant Pleural Mesothelioma by Modulating HAS3 Expression

Increased hyaluronic acid (HA) production is often associated with cancer progression. In malignant pleural mesothelioma (MPM), HA is found at elevated levels in pleural effusions and sera of patients, and it has been widely debated whether MPM cells are able to produce HA by themselves or through the release of growth factors stimulating other cells. Another key component of the MPM microenvironment is C1q, which can act as a pro-tumorigenic factor favoring cell adhesion, migration and proliferation. The aim of the current study was to prove that MPM primary cells are able to synthesize HA and to inquire the stimulus given by C1q–HA matrix to HA synthesis. We confirmed the presence of a HA coat and cable-like structures around MPM primary cells, as well as an intracellular pool, mainly localized in the cytoplasmic and perinuclear region. After evaluating HA synthase (HAS) enzymes’ basal expression in MPM primary cells, we found that C1q bound to HA was able to impinge upon HA homeostasis by upregulating HAS3 both at the mRNA and the protein levels. High expression of HAS3 has been correlated with a shorter life expectancy in MPM by bioinformatical analysis. These data confirmed that C1q bound to HA may exert pro-tumorigenic activity and identified HAS3 as a potential target in MPM.

Inhibitor of Hyaluronic Acid Synthesis 4-Methylumbelliferone as an Anti-Inflammatory Modulator of LPS-Mediated Astrocyte Responses

Astrocytes are glial cells that play an important role in neuroinflammation. Astrocytes respond to many pro-inflammatory stimuli, including lipopolysaccharide (LPS), an agonist of Toll-like receptor 4 (TLR4). Regulatory specificities of inflammatory signaling pathways are still largely unknown due to the ectodermal origin of astrocytes. Recently, we have shown that hyaluronic acid (HA) may form part of astrocyte inflammatory responses. Therefore, we tested 4-methylumbelliferone (4-MU), a specific inhibitor of HA synthesis, as a possible regulator of LPS-mediated responses. Rat primary astrocytes were treated with LPS with and without 4-MU and gene expression levels of inflammatory (interleukins 1β, (IL-1β), 6, (IL-6), tumor necrosis factor alpha TNFα,) and resolution interleukin 10 (IL-10) markers were evaluated via real-time PCR and western blot. The release of cytokines and HA was determined by ELISA. Oxylipin profiles were measured by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis. Our data show that 4-MU (i) has anti-inflammatory effects in the course of TLR4 activation, decreasing the cytokines level TNFα, IL-6 and IL-1β and increasing IL-10, (ii) downregulates prostaglandin synthesis but not via cyclooxygenases COX-1 and COX-2 pathways, (iii) modulates HA synthesis and decreases LPS-induced HA synthase mRNA expression (HAS-1, HAS-2) but does not have an influence on HAS-3, HYAL1 and HYAL2 mRNAs; (iv) the effects of 4-MU are predominantly revealed via JNK but not p38, ERK mitogen-activated protein kinases (MAPKs) or nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) pathways. For the first time, it is shown that 4-MU possesses the useful potential to regulate an inflammatory astrocyte response.

CircHYBID Regulates Hyaluronan Metabolism in Chondrocytes by Promoting the Expression of the hsa-miR-29b-3p Target Gene TGF-β1

Background: Hyaluronan (HA) metabolism by chondrocytes is essential for cartilage development and homeostasis. However, the function of circular RNAs (circRNAs)in HA metabolism is limited. In this study, we profiled the role of the novel HA-related circRNA circHYBID in the progression of osteoarthritis.Methods: The function of circHYBID in HA metabolism in chondrocytes was investigated using gain-of-function experiments. The mechanism of HA-binding protein involved in hyaluronan depolymerization (HYBID) was confirmed through bioinformatics analysis and luciferase assays. The expression correlation of the circHYBID–hsa-miR-29b-3p–transforming growth factor (TGF)-β1 axis was examined by qRT-PCR and western blotting. CircHYBID, TGF-β1, and HA levels in cartilage samples were evaluated using immunohistochemistry. ELISA assay was used to assess HA accumulation in chondrocyte supernatant.Results: CircHYBID expression was significantly downregulated in damaged cartilage samples comparing with that in the corresponding intact cartilage samples. CircHYBID expression was inversely correlated with the Mankin score and positively correlated with HA expression. Interleukin-1β stimulation in chondrocytes downregulated circHYBID expression and decreased HA accumulation. Gain-of-function experiments revealed that circHYBID overexpression in chondrocytes increased HA accumulation by regulating HA synthase 2 and HYBID expression. Further mechanism analysis illustrated that circHYBID upregulated TGF-β1 expression by sponging hsa-miR-29b-3p. Conclusions: Our results describe a novel HA-related circRNA that could promote the synthesis and accumulation of HA. The circHYBID–hsa-miR-29b-3p–TGF-β1 axis may play a powerful regulatory role in HA metabolism and Osteoarthritis (OA) progression. Thus, these findings will provide new perspectives for studies on OA pathogenesis, and circHYBID may serve as a potential target for OA therapy.

Slit2 Regulates Hyaluronan & Cytokine Synthesis in Fibrocytes: Potential Relevance to Thyroid-Associated Ophthalmopathy

Context CD34+ fibrocytes have been implicated in development of thyroid-associated ophthalmopathy (TAO), a consequential autoimmune manifestation of Graves disease (GD). In TAO, CD34+ fibrocytes appear to masquerade as CD34+ orbital fibroblasts mixed with CD34- OF (collectively, GD-OF). Slit2, an axon guidance glycoprotein, is expressed by CD34- OF and attenuates GD-OF gene expression. Cardinal features of TAO include hyaluronan (HA) accumulation and cytokine-driven inflammation. Objective Compare expression of HA synthase isoenzymes (HAS1-3), UDP-glucose dehydrogenase (UGDH), synthesis of HA, interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) in fibrocytes and GD-OF. Determine whether Slit2 alters gene expression patterns. Design/Setting/Participants Patients with TAO and healthy donors were recruited from an academic practice. Main Outcome Measures Real-time polymerase chain reaction, HA, IL-6, and TNF-α immunoassays. Results HA synthesis and release from fibrocytes is substantially lower than in GD-OF. HAS1 expression dominates in fibrocytes while HAS2 in GD-OF. In contrast, HAS2 and UGDH expression dominate GD-OF and localize to CD34- OF. Recombinant human Slit2 (rhSlit2) substantially upregulates HA synthesis and HAS2 expression in fibrocytes but attenuates IL-6 and TNF-α production in these cells. In contrast, knocking down Slit2 in GD-OF reduces HA synthesis and HAS2 and UGDH expression while upregulating IL-6 and TNF-α. Conclusion The dramatic differences in HA, IL-6, and TNF-α production, and HAS and UGDH expression found in fibrocytes and GD-OF appear, at least in part, to be attributable to Slit2. These findings provide novel insight into the differences in gene expression exhibited by CD34+ fibrocytes and CD34+ OF and therefore reveal important aspects of disease pathogenesis.

Knockdown Of Udp-Glucose Dehydrogenase Facilitates Epirubicin-Resistance In MDA-MB-231 Breast Cancer Cells By Regulation Of Hyaluronan Synthesis.

Background: The catalytic action of the UDP-glucose transferase dehydrogenase (UGDH) enzyme produces UDP-glucuronic acid (UDP-GlcUA). The main detoxifying pathway of Epirubicin (EPI) is via glucuronidation by the specific transferase UGT2B7 (UDP-Glucuronosyltransferase-2B7), adding UDP-GlcUA to generates 4´-O-b-D-glucuronyl-4´-epi-doxorubicin. UDP-GlcUA is also a precursor of several glycosaminoglycans (GAGs) like hyaluronan (HA). Therefore, the EPI detoxifying pathway might be associated with GAGs metabolism, related to drug deactivation and tumor resistance. This work aimed to evaluate the effect of knockdown on the UGDH gene on EPI response and HA metabolism in the aggressive breast cancer cells MDA-MB 231. Methods: MDA-MB-231 cells were transfected in vitro with UGDH-specific siRNA for UGDH knockdown and treated with EPI. Viability, apoptosis, and cytotoxicity effects were evaluated. EPI intracellular accumulation was analyzed by flow cytometry. Differences in extracellular matrix (ECM) were analyzed through a particle exclusion assay and the composition of HA using hyaluronidase. The soluble HA secreted was detected by an ELISA like assay. Besides, gene expression of HA synthase and hyaluronidase (HYAL) enzymes were analyzed by RT-qPCR. To analyze the effect of UGDH knockdown and EPI treatment on autophagy, we evaluated the expression of the autophagosome marker, LC3-II by RT-qPCR and western blot, and its subcellular localization by confocal microscopy. Results: EPI accumulation increased during UGDH knockdown, but it was observed a decrease in cell death. HA synthesis and HA coated around the cells increased. In turn, it was found up-regulation of the expression of HYALs. Within the mechanisms activated by tumor cells to avoid EPI activity, an increase in autophagy was detected. Conclusions: for the first time we show that an increase in the expression, deposition and catabolism of HA positively contributed to the development of a resistant phenotype in breast cancer cells by a mechanism associated to sugar metabolism, specifically of UDP-GlcUA .

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.

Heterologous Hyaluronic Acid Production in Kluyveromyces lactis

Hyaluronic Acid (HA) is a biopolymer composed by the monomers Glucuronic Acid (GlcUA) and N-Acetyl Glucosamine (GlcNAc). It has a broad range of applications in the field of medicine, being marketed between USD 1000–5000/kg. Its primary sources include extraction of animal tissue and fermentation using pathogenic bacteria. However, in both cases, extensive purification protocols are required to prevent toxin contamination. In this study, aiming at creating a safe HA producing microorganism, the generally regarded as safe (GRAS) yeast Kluyveroymyces lactis is utilized. Initially, the hasB (UDP-Glucose dehydrogenase) gene from Xenopus laevis (xlhasB) is inserted. After that, four strains are constructed harboring different hasA (HA Synthase) genes, three of humans (hshasA1, hshasA2, and hshasA3) and one with the bacteria Pasteurella multocida (pmhasA). Transcript values analysis confirms the presence of hasA genes only in three strains. HA production is verified by scanning electron microscopy in the strain containing the pmHAS isoform. The pmHAS strain is grown in a 1.3 l bioreactor operating in a batch mode, the maximum HA levels are 1.89 g/L with a molecular weight of 2.097 MDa. This is the first study that reports HA production in K. lactis and it has the highest HA titers reported among yeast.