Unravelling the role of HAS2, GREM1, and PTGS2 gene expression in cumulus cells: implications for human oocyte development competency - a systematic review and integrated bioinformatic analysis.

The leading indicator for successful outcomes in in-vitro fertilization (IVF) is the quality of gametes in oocytes and sperm. Thus, advanced research aims to highlight the parameter in assessing these qualities - DNA fragmentation in sperm and oocyte development capacity (ODC) via evaluation of microenvironments involving its maturation process. Regarding oocytes, most evidence reveals the role of cumulus cells as non-invasive methods in assessing their development competency, mainly via gene expression evaluation. Our review aims to consolidate the evidence of GDF-9 derivatives, the HAS2, GREM1, and PTGS2 gene expression in cumulus cells used as ODC markers in relevant publications and tailored to current IVF outcomes. In addition to that, we also added the bioinformatic analysis in our review to strengthen the evidence aiming for a better understanding of the pathways and cluster of the genes of interest - HAS2, GREM1, and PTGS2 in cumulus cell level. Otherwise, the current non-invasive method can be used in exploring various causes of infertility that may affect these gene expressions at the cumulus cell level. Nevertheless, this method can also be used in assessing the ODC in various cohorts of women or as an improvement of markers following targeted tools or procedures by evaluating the advancement of these gene expressions following the targeted intervention.

Berberine alleviates inflammation in polycystic ovary syndrome by inhibiting hyaluronan synthase 2 expression.

BACKGROUND: Polycystic ovary syndrome (PCOS) is a heterogeneous metabolic and endocrine disorder that causes anovulatory infertility and abnormal folliculogenesis in women of reproductive age. Several studies have revealed inflammation in PCOS follicles, and recent evidence suggests that Berberine (BBR) effectively reduces inflammatory responses in PCOS, however, the underlying mechanisms remain unclear. PURPOSE: To determine the underlying mechanisms by which BBR alleviates inflammation in PCOS. STUDY DESIGN: Primary human GCs from healthy women and women with PCOS, and KGN cells were used for in vitro studies. ICR mice were used for in vivo studies. METHODS: Gene expression was measured using RT-qPCR. HAS2, inflammatory cytokines, and serum hormones were assayed by ELISA. Protein expression profiles were assayed by Western blot. Chronic low-grade inflammatory mouse models were developed by intraperitoneal injection with LPS, and PCOS mouse models were established by subcutaneous intraperitoneal injection of DHEA. BBR and 4-MU were administered by gavage. Ovarian morphologic changes were evaluated using H&E staining. HAS2 expression in the ovary was assayed using Western blot and immunohistochemistry. RESULTS: Our results confirmed that HAS2 expression and hyaluronan (HA) accumulation are closely associated with inflammatory responses in PCOS. Data obtained from in vitro studies showed that HAS2 and inflammatory genes (e.g., MCP-1, IL-1ß, and IL-6) are significantly upregulated in PCOS samples and LPS-induced KGN cells compared to their control groups. In addition, these effects were reversed by blocking HAS2 expression or HA synthesis using BBR or 4-MU, respectively. Furthermore, HAS2 overexpression induces the expression of inflammatory genes in PCOS. These results were further confirmed in LPS- and DHEA-induced mouse models, where inflammatory genes were reduced by BBR or 4-MU, and ovarian morphology was restored. CONCLUSIONS: Our results define previously unknown links between HAS2 and chronic low-grade inflammation in the follicles of women with PCOS. BBR exerts its anti-inflammatory effects by down-regulating HAS2. This study provides a novel therapeutic target for alleviating ovarian inflammation in women with PCOS.

AßPP-tau-HAS1 axis trigger HAS1-related nuclear speckles and gene transcription in Alzheimer's disease.

As the backbone of the extracellular matrix (ECM) and the perineuronal nets (PNNs), hyaluronic acid (HA) provides binding sites for proteoglycans and other ECM components. Although the pivotal of HA has been recognized in Alzheimer's disease (AD), few studies have addressed the relationship between AD pathology and HA synthases (HASs). Here, HASs in different regions of AD brains were screened in transcriptomic database and validated in AßPP/PS1 mice. We found that HAS1 was distributed along the axon and nucleus. Its transcripts were reduced in AD patients and AßPP/PS1 mice. Phosphorylated tau (p-tau) mediates AßPP-induced cytosolic-nuclear translocation of HAS1, and negatively regulated the stability, monoubiquitination, and oligomerization of HAS1, thus reduced the synthesis and release of HA. Furthermore, non-ubiquitinated HAS1 mutant lost its enzyme activity, and translocated from the cytosol into the nucleus, forming nuclear speckles (NS). Unlike the splicing-related NS, less than 1 % of the non-ubiquitinated HAS1 co-localized with SRRM2, proving the regulatory role of HAS1 in gene transcription, indirectly. Thus, differentially expressed genes (DEGs) related to both non-ubiquitinated HAS1 mutant and AD were screened using transcriptomic datasets. Thirty-nine DEGs were identified, with 64.1 % (25/39) showing consistent results in both datasets. Together, we unearthed an important function of the AßPP-p-tau-HAS1 axis in microenvironment remodeling and gene transcription during AD progression, involving the ubiquitin-proteasome, lysosome, and NS systems.

Knockdown of Hyaluronan synthase 2 suppresses liver fibrosis in mice via induction of transcriptomic changes similar to 4MU treatment.

Hepatic fibrosis remains a significant clinical challenge due to ineffective treatments. 4-methylumbelliferone (4MU), a hyaluronic acid (HA) synthesis inhibitor, has proven safe in phase one clinical trials. In this study, we aimed to ameliorate liver fibrosis by inhibiting HA synthesis. We compared two groups of mice with CCl4-induced fibrosis, treated with 4-methylumbelliferone (4MU) and hyaluronan synthase 2 (HAS2) targeting siRNA (siHAS2). The administration of 4MU and siHAS2 significantly reduced collagen and HA deposition, as well as biochemical markers of hepatic damage induced by repeated CCl4 injections. The transcriptomic analysis revealed converging pathways associated with downstream HA signalling. 4MU- and siHAS2-treated fibrotic livers shared 405 upregulated and 628 downregulated genes. These genes were associated with xenobiotic and cholesterol metabolism, mitosis, endoplasmic reticulum stress, RNA processing, and myeloid cell migration. The functional annotation of differentially expressed genes (DEGs) in siHAS2-treated mice revealed attenuation of extracellular matrix-associated pathways. In comparison, in the 4MU-treated group, DEGs were related to lipid and bile metabolism pathways and cell cycle. These findings confirm that HAS2 is an important pharmacological target for suppressing hepatic fibrosis using siRNA.

Melatonin inhibits the stemness of head and neck squamous cell carcinoma by modulating HA synthesis via the FOSL1/HAS3 axis.

Hyaluronic acid (HA) is a glycosaminoglycan and the main component of the extracellular matrix (ECM), which has been reported to interact with its receptor CD44 to play critical roles in the self-renewal and maintenance of cancer stem cells (CSCs) of multiple malignancies. Melatonin is a neuroendocrine hormone with pleiotropic antitumor properties. However, whether melatonin could regulate HA accumulation in the ECM to modulate the stemness of head and neck squamous cell carcinoma (HNSCC) remains unknown. In this study, we found that melatonin suppressed CSC-related markers, such as CD44, of HNSCC cells and decreased the tumor-initiating frequency of CSCs in vivo. In addition, melatonin modulated HA synthesis of HNSCC cells by downregulating the expression of hyaluronan synthase 3 (HAS3). Further study showed that the Fos-like 1 (FOSL1)/HAS3 axis mediated the inhibitory effects of melatonin on HA accumulation and stemness of HNSCC in a receptor-independent manner. Taken together, melatonin modulated HA synthesis through the FOSL1/HAS3 axis to inhibit the stemness of HNSCC cells, which elucidates the effect of melatonin on the ECM and provides a novel perspective on melatonin in HNSCC treatment.

Naked mole-rat hyaluronan.

Naked mole rats (NMRs) are renowned for their exceptional longevity and remarkable maintenance of health throughout their lifetime. Their subterranean lifestyle has led to adaptations that have resulted in elevated levels of a very high molecular weight hyaluronan in their tissues. Hyaluronan, a glycosaminoglycan, is a key component of the extracellular matrix, which plays a critical role in maintaining tissue structure and regulating cell signaling pathways. This phenomenon in NMRs is attributed to a higher processing and production capacity by some of their hyaluronan synthases, along with lower degradation by certain hyaluronidases. Furthermore, this adaptation indirectly confers several advantages to NMRs, such as the preservation of skin elasticity and youthful appearance, accelerated wound healing, protection against oxidative stress, and resistance to conditions such as cancer and arthritis, largely attributable to CD44 signaling and other intricate mechanisms. Thus, the main objective of this study was to conduct a comprehensive study of the distinctive features of NMR hyaluronan, particularly emphasizing the currently known molecular mechanisms that contribute to its beneficial properties. Furthermore, this research delves into the potential applications of NMR hyaluronan in both cosmetic and therapeutic fields, as well as the challenges involved.

Mesenchymal stromal cells confer breast cancer doxorubicin resistance by producing hyaluronan.

Chemotherapy resistance represents a major cause of therapeutic failure and mortality in cancer patients. Mesenchymal stromal cells (MSCs), an integral component of tumor microenvironment, are known to promote drug resistance. However, the detailed mechanisms remain to be elucidated. Here, we found that MSCs confer breast cancer resistance to doxorubicin by diminishing its intratumoral accumulation. Hyaluronan (HA), a major extracellular matrix (ECM) product of MSCs, was found to mediate the chemoresistant effect. The chemoresistant effect of MSCs was abrogated when hyaluronic acid synthase 2 (HAS2) was depleted or inhibited. Exogenous HA also protected tumor grafts from doxorubicin. Molecular dynamics simulation analysis indicates that HA can bind with doxorubicin, mainly via hydrophobic and hydrogen bonds, and thus reduce its entry into breast cancer cells. This mechanism is distinct from the reported chemoresistant effect of HA via its receptor on cell surface. High HA serum levels were also found to be positively associated with chemoresistance in breast cancer patients. Our findings indicate that the HA-doxorubicin binding dynamics can confer cancer cells chemoresistance. Reducing HA may enhance chemotherapy efficacy.

Increased hyaluronan by naked mole-rat Has2 improves healthspan in mice.

Abundant high-molecular-mass hyaluronic acid (HMM-HA) contributes to cancer resistance and possibly to the longevity of the longest-lived rodent-the naked mole-rat1,2. To study whether the benefits of HMM-HA could be transferred to other animal species, we generated a transgenic mouse overexpressing naked mole-rat hyaluronic acid synthase 2 gene (nmrHas2). nmrHas2 mice showed an increase in hyaluronan levels in several tissues, and a lower incidence of spontaneous and induced cancer, extended lifespan and improved healthspan. The transcriptome signature of nmrHas2 mice shifted towards that of longer-lived species. The most notable change observed in nmrHas2 mice was attenuated inflammation across multiple tissues. HMM-HA reduced inflammation through several pathways, including a direct immunoregulatory effect on immune cells, protection from oxidative stress and improved gut barrier function during ageing. These beneficial effects were conferred by HMM-HA and were not specific to the nmrHas2 gene. These findings demonstrate that the longevity mechanism that evolved in the naked mole-rat can be exported to other species, and open new paths for using HMM-HA to improve lifespan and healthspan.

Hyaluronan Metabolism in Urologic Cancers.

Hyaluronan (HA) is one of the major components of the extracellular matrix in tumor tissue. Recent reports have made it clear that the balance of HA synthesis and degradation is critical for tumor progression. HA is synthesized on the cytoplasmic surface of the plasma membrane by hyaluronan synthases (HAS) and extruded into the extracellular space. Excessive HA production in cancer is associated with enhanced HA degradation in the tumor microenvironment, leading to the accumulation of HA fragments with small molecular weight. These perturbations in both HA synthesis and degradation may play important roles in tumor progression. Recently, it has become increasingly clear that small HA fragments can induce a variety of biological events, such as angiogenesis, cancer-promoting inflammation, and tumor-associated immune suppression. Progression of urologic malignancies, particularly of prostate and bladder cancers, as well as of certain types of kidney cancer show markedly perturbed metabolism of tumor-associated HA. This review highlights the recent research findings regarding HA metabolism in tumor microenvironments with a special focus on urologic cancers. It also will discuss the potential implications of these findings for the development of novel therapeutic interventions for the treatment of prostate, bladder, and kidney cancers.

Mimicking Antioxidases and Hyaluronan Synthase: A Zwitterionic Nanozyme for Photothermal Therapy of Osteoarthritis.

Restoring joint homeostasis is crucial for relieving osteoarthritis (OA). Current strategies are limited to unilateral efforts in joint lubrication, inhibition of inflammation, free radicals scavenging, and cartilage regeneration. Herein, by modifying molybdenum disulfide (MoS2 ) with Mg2+ -doped polydopamine and coating with polysulfobetaines, a dual-bionic photothermal nanozyme (MPMP) is constructed to mimic antioxidases/hyaluronan synthase for OA therapy. Photothermally enhanced lubrication lowers the coefficient of friction (0.028) in the early stage of OA treatment. The antioxidases-mimicking properties of MPMP nanozyme contribute to eliminating reactive oxygen and nitrogen species (ROS/RNS) (over 90% of scavenging ratio for H2 O2 /·OH/O· 2 - /DPPH/ABTS+ ) and supplying O2 . With NIR irradiation, the MPMP nanozyme triggers thermogenesis (upregulating HSP70 expression) and Mg2+ release, which promotes the chondrogenesis in inflammatory conditions by deactivating NF-κB/IL-17 signaling pathways and enhancing MAPK signaling pathway. Benefiting from HSP70 and Mg2+ , MPMP-NIR shows HAS-mimicking activity to increase the intracellular (twofold) and extracellular (3.12-fold) HA production. Therefore, MPMP-NIR demonstrates superior spatiotemporally therapeutic effect on OA in mice model, in terms of osteophytes (83.41% of reduction), OARSI scores (88.57% of reduction), and ACAN expression (2.70-fold of increment). Hence, insights into dual-bionic nanozymes can be a promising strategy for OA therapy or other inflammation-related diseases.

Downregulation of HAS­2 regulates the chondrocyte cytoskeleton and induces cartilage degeneration by activating the RhoA/ROCK signaling pathway.

Osteoarthritis (OA) is a progressive joint disorder, which is principally characterized by the degeneration and destruction of articular cartilage. The cytoskeleton is a vital structure that maintains the morphology and function of chondrocytes, and its destruction is a crucial risk factor leading to chondrocyte degeneration and OA. Hyaluronan synthase­2 (HAS­2) is a key enzyme in synthesizing hyaluronic acid (HA) in vivo. The synthesis of high molecular weight HA catalyzed by HAS­2 serves a vital role in joint movement and homeostasis; however, it is unclear what important role HAS­2 plays in maintaining chondrocyte cytoskeleton morphology and in cartilage degeneration. The present study downregulated the expression of HAS­2 by employing 4­methylumbelliferone (4­MU) and RNA interference. In vitro experiments, including reverse transcription­quantitative PCR, western blotting, laser scanning confocal microscopy and flow cytometry were subsequently performed. The results revealed that downregulation of HAS­2 could activate the RhoA/ROCK signaling pathway, cause morphological abnormalities, decrease expression of the chondrocyte cytoskeleton proteins and promote chondrocyte apoptosis. In vivo experiments, including immunohistochemistry and Mankin's scoring, were performed to verify the effect of HAS­2 on the chondrocyte cytoskeleton, and it was revealed that inhibition of HAS­2 could cause cartilage degeneration. In conclusion, the present results revealed that downregulation of HAS­2 could activate the RhoA/ROCK pathway, cause abnormal morphology and decrease chondrocyte cytoskeleton protein expression, leading to changes in the signal transduction and biomechanical properties of chondrocytes, promotion of chondrocyte apoptosis and the induction of cartilage degeneration. Moreover, the clinical application of 4­MU may cause cartilage degeneration. Therefore, targeting HAS­2 may provide a novel therapeutic strategy for delaying chondrocyte degeneration, and the early prevention and treatment of OA.

MicroRNA-376b is involved in the pathogenesis of thyroid-associated ophthalmopathy by regulating HAS2.

PURPOSE: The aim of this study was to investigate the microRNA (miRNA) expression profile in peripheral blood mononuclear cells (PBMC) of thyroid-associated ophthalmopathy (TAO) patients and to explore the molecular mechanisms of MicroRNA-376b (miR-376b) in the pathogenesis of TAO. METHODS: PBMCs from TAO patients and healthy controls were analyzed by miRNA microarray to screen for the significantly differentially expressed miRNAs. The miR-376b expression in PBMCs were confirmed by quantitative real-time polymerase chain reaction (qRT-PCR). The downstream target of miR-376b was screened by online bioinformatics, and detected by qRT-PCR and Western blotting. RESULTS: Compared with normal controls, 26 miRNAs were significantly different in PBMCs of TAO patients (14 miRNAs were down-regulated and 12 miRNAs were up-regulated). Among them, miR-376b expression was significantly decreased in PBMCs from TAO patients compared to healthy controls. Spearman correlation analysis revealed that miR-376b expression in PBMCs was significantly negatively correlated with free triiodothyronine (FT3), and positively correlated with thyroid-stimulating hormone (TSH). MiR-376b expression was obviously reduced in 6T-CEM cells after triiodothyronine (T3) stimulation compared to controls. MiR-376b mimics significantly decreased hyaluronan synthase 2 (HAS2) protein expression and the mRNA expression of intercellular cell adhesion molecule-1 (ICAM1) and tumor necrosis factor-α (TNF-α) in 6T-CEM cells, whereas miR-376b inhibitors markedly elevated HAS2 protein expression and gene expression of ICAM1 and TNF-α. CONCLUSIONS: MiR-376b expression in PBMCs was significantly decreased in PBMCs from TAO patients compared with the healthy controls. MiR-376b, regulated by T3, could modulate the expression of HAS2 and inflammatory factors. We speculate that miR-376b may be involved in the pathogenesis of TAO patients by regulating the expression of HAS2 and inflammatory factors.

Particle Exclusion Assay: A Tool for Measuring Hyaluronan Pericellular Matrix.

Hyaluronan (HA) is the most abundant glycosaminoglycan in the extracellular matrix, and its deposition is strictly related to changes in cellular behaviors, such as cell migration, proliferation, and adhesion. Pericellular HA is abundant in a variety of cell types, and its amount could reflect specific conditions, thus suggesting a particular cellular status.Particle exclusion assay is a useful tool to visualize pericellular matrices with a high HA content, simply employing microscope image analysis. This approach is quick and allows to visualize the presence of a clear pericellular region around single cells, where fixed red blood cells are excluded if the pericellular matrix has been deposited.

Hyaluronan nanoscale clustering and Hyaluronan synthase 2 expression are linked to the invasion of child fibroblasts and infantile fibrosarcoma in vitro and in vivo.

Infantile fibrosarcoma is a rare childhood tumour that originates in the fibrous connective tissue of the long bones for which there is an urgent need to identify novel therapeutic targets. This study aims to clarify the role of the extracellular matrix component hyaluronan in the invasion of child fibroblasts and Infantile fibrosarcoma into the surrounding environment. Using nanoscale super-resolution STED (Stimulated emission depletion) microscopy followed by computational image analysis, we observed, for the first time, that invasive child fibroblasts showed increased nanoscale clustering of hyaluronan at the cell periphery, as compared to control cells. Hyaluronan was not observed within focal adhesions. Bioinformatic analyses further revealed that the increased nanoscale hyaluronan clustering was accompanied by increased gene expression of Hyaluronan synthase 2, reduced expression of Hyaluronidase 2 and CD44, and no change of Hyaluronan synthase 1 and Hyaluronidases 1, 3, 4 or 5. We further observed that the expression of the Hyaluronan synthase 1, 2 and 3, and the Hyaluronidase 3 and 5 genes was linked to reduced life expectancy of fibrosarcoma patients. The invasive front of infantile fibrosarcoma tumours further showed increased levels of hyaluronan, as compared to the tumour centre. Taken together, our findings are consistent with the possibility that while Hyaluronan synthase 2 increases the levels, the Hyaluronidases 3 and 5 reduce the weight of hyaluronan, resulting in the nanoscale clustering of hyaluronan at the leading edge of cells, cell invasion and the spread of Infantile fibrosarcoma.

Transforming Growth Factor-ß Receptor-Mediated, p38 Mitogen-Activated Protein Kinase-Dependent Signaling Drives Enhanced Myofibroblast Differentiation during Skin Wound Healing in Mice Lacking Hyaluronan Synthases 1 and 3.

Normal myofibroblast differentiation is critical for proper skin wound healing. Neoexpression of α-smooth muscle actin (α-SMA), a marker for myofibroblast differentiation, is driven by transforming growth factor (TGF)-ß receptor-mediated signaling. Hyaluronan and its three synthesizing enzymes, hyaluronan synthases (Has 1, 2, and 3), also participate in this process. Closure of skin wounds is significantly accelerated in Has1/3 double-knockout (Has1/3-null) mice. Herein, TGF-ß activity and dermal collagen maturation were increased in Has1/3-null healing skin. Cultures of primary skin fibroblasts isolated from Has1/3-null mice had higher levels of TGF-ß activity, α-SMA expression, and phosphorylation of p38 mitogen-activated protein kinase at Thr180/Tyr182, compared with wild-type fibroblasts. p38α mitogen-activated protein kinase was a necessary element in a noncanonical TGF-ß receptor signaling pathway driving α-SMA expression in Has1/3-null fibroblasts. Myocardin-related transcription factor (MRTF), a cofactor that binds to the transcription factor serum response factor (SRF), was also critical. Nuclear localization of MRTF was increased, and MRTF binding to SRF was enhanced in Has1/3-null fibroblasts. Inhibition of MRTF or SRF expression by RNA interference suppresses α-SMA expression at baseline and diminished its overexpression in Has1/3-null fibroblasts. Interestingly, total matrix metalloproteinase activity was increased in healing skin and fibroblasts from Has1/3-null mice, possibly explaining the increased TGF-ß activation.

The Upregulation of HAS2-AS1 Relates to the Granulosa Cell Dysfunction by Repressing TGF-ß Signaling and Upregulating HAS2.

HAS2 antisense RNA 1 (HAS2-AS1) is a long noncoding RNA that has increased expression in mature granulosa cells (GCs) and contributes to cumulus expansion by regulating HAS2 expression. However, the roles of HAS2-AS1 during the pathological process of polycystic ovary syndrome (PCOS) are still unclear. This study investigated the roles of HAS2-AS1 in patients with PCOS. Here, a significant upregulation of HAS2-AS1 was found in the primary GCs from patients with PCOS, which was positively correlated with the level of the protein HAS2. The knockdown of HAS2 restored the upregulation of HAS2-AS1 in promoting migration but could not restore the effects of HAS2-AS1 overexpression in promoting proliferation and repressing apoptosis. Transforming growth factor ß (TGF-ß) upregulated HAS2-AS1 levels, while HAS2-AS1 functioned as a feedback inhibition factor repressing TGF-ß signaling by inhibiting TGF-ß receptor type 2 (TGFBR2) expression. HAS2-AS1 bonded with EZH2 and guided the polycomb complex 2 to the TGFBR2 promoter region. HAS2-AS1 overexpression induced H3K27 hypermethylation in the TGFBR2 promoter region and then repressed TGFBR2 transcription in KGN cells and primary GCs. In conclusion, we identified for the first time that HAS2-AS1 is upregulated in patients with PCOS and represses TGF-ß signaling via inducing TGFBR2 promoter region hypermethylation, which allowed us to explore the pathological processes of PCOS.

Hyaluronan synthase 3 is protective after cardiac ischemia-reperfusion by preserving the T cell response.

Dysregulated extracellular matrix (ECM) is a hallmark of adverse cardiac remodeling after myocardial infarction (MI). Previous work from our laboratory suggests that synthesis of the major ECM component hyaluronan (HA) may be beneficial for post-infarct healing. Here, we aimed to investigate the mechanisms of hyaluronan synthase 3 (HAS3) in cardiac healing after MI. Mice with genetic deletion of Has3 (Has3 KO) and wildtype mice (WT) underwent 45 min of ischemia with subsequent reperfusion (I/R), followed by monitoring of heart function and analysis of tissue remodeling for up to three weeks. Has3 KO mice exhibited impaired cardiac function as evidenced by a reduced ejection fraction. Accordingly, Has3 deficiency also resulted in an increased scar size. Cardiac fibroblast activation and CD68+ macrophage counts were similar between genotypes. However, we found a significant decrease in CD4 T cells in the hearts of Has3 KO mice seven days post-MI, in particular reduced numbers of CD4+CXCR3+ Th1 and CD4+CD25+Treg cells. Furthermore, Has3 deficient cardiac T cells were less activated and more apoptotic as shown by decreased CD69+ and increased annexin V+ cells, respectively. In vitro assays using activated splenic CD3 T cells demonstrated that Has3 deficiency resulted in reduced expression of the main HA receptor CD44 and diminished T cell proliferation. T cell transendothelial migration was similar between genotypes. Of note, analysis of peripheral blood from patients with ST-elevation myocardial infarction (STEMI) revealed that HAS3 is the predominant HAS isoenzyme also in human T cells. In conclusion, our data suggest that HAS3 is required for mounting a physiological T cell response after MI to support cardiac healing. Therefore, our study may serve as a foundation for the development of novel strategies targeting HA-matrix to preserve T cell function after MI.

Hyaluronan synthase 2, a target of miR-200c, promotes carbon tetrachloride-induced acute and chronic liver inflammation via regulation of CCL3 and CCL4.

Liver fibrosis occurs during wound healing after repeated liver injury and is characterized by extensive extracellular matrix deposition. We previously identified hyaluronan synthase 2 (HAS2) as a driver of liver fibrosis and hepatic stellate cell (HSC) activation. Developing strategies to suppress HSC activation is key to alleviating liver fibrosis, and HAS2 is an attractive candidate for intervention. To gain insight into the molecular function of HAS2, we investigated its posttranscriptional regulation. We found that miR-200c directly targets the 3' untranslated regions of HAS2. Moreover, miR-200c and HAS2 were inversely expressed in fibrotic human and mouse livers. After establishing the direct interaction between miR-200c and HAS2, we investigated the functional outcome of regulating HAS2 expression in three murine models: CCl4-induced acute liver injury, CCl4-induced chronic liver fibrosis, and bile duct ligation-induced liver fibrosis. Hepatic Has2 expression was induced by acute and chronic CCl4 treatment. In contrast, miR-200c expression was decreased after CCl4 treatment. HSC-specific Has2 deletion reduced the expression of inflammatory markers and infiltration of macrophages in the models. Importantly, hyaluronidase-2 (HYAL2) but not HYAL1 was overexpressed in fibrotic human and murine livers. HYAL2 is an enzyme that can cleave the extracellular matrix component hyaluronan. We found that low-molecular-weight hyaluronan stimulated the expression of inflammatory genes. Treatment with the HA synthesis inhibitor 4-methylumbelliferone alleviated bile duct ligation-induced expression of these inflammatory markers. Collectively, our results suggest that HAS2 is negatively regulated by miR-200c and contributes to the development of acute liver injury and chronic liver inflammation via hyaluronan-mediated immune signaling.

Cell-Type-Specific Expression of Hyaluronan Synthases HAS2 and HAS3 Promotes Goblet Cell Hyperplasia in Allergic Airway Inflammation.

Allergic rhinitis (AR) is a multifactorial airway disease characterized by basal and goblet cell hyperplasia. Hyaluronic acid (HA) is a major component of extracellular matrix and a critical contributor to tissue repair and remodeling after injury. We previously demonstrated that the intermediate progenitor cell (IPC) surface marker CD44v3 is upregulated in the basal and suprabasal layers of well-differentiated primary human nasal epithelial (HNE) cells after stimulation with the Th2 (T-helper cell type 2) cytokine IL-4, and an antibody blocking the CD44v3-HA interaction suppressed IL-4-induced goblet cell hyperplasia. We now show that the expression of HA and two HA synthases, HAS2 and HAS3, was upregulated in both the nasal surface epithelium of subjects with AR and IL-4-stimulated HNE cells. Inhibition of HA synthesis by 4-methylumbelliferone suppressed IL-4-induced goblet cell hyperplasia. Moreover, HAS2 and HAS3 were expressed in IPCs depending on the differentiation events, as follows: the rapid, transient upregulation of HAS2 induced basal IPC proliferation and basal-to-suprabasal transition, whereas the delayed upregulation of HAS3 promoted the transition of suprabasal IPCs to a goblet cell fate. 4-methylumbelliferone treatment in a house dust mite-induced murine AR model attenuated goblet cell metaplasia. Last, HA concentrations in nasal epithelial lining fluids from patients with AR positively correlated with the concentrations of mediators causing allergic inflammation. These data suggest that HA produced after the sequential upregulation of HAS2 and HAS3 contributes to goblet cell hyperplasia in allergic airway inflammation and modulates disease progression.

3'UTR shortening of HAS2 promotes hyaluronan hyper-synthesis and bioenergetic dysfunction in pulmonary hypertension.

Pulmonary hypertension (PH) comprises a diverse group of disorders that share a common pathway of pulmonary vascular remodeling leading to right ventricular failure. Development of anti-remodeling strategies is an emerging frontier in PH therapeutics that requires a greater understanding of the interactions between vascular wall cells and their extracellular matrices. The ubiquitous matrix glycan, hyaluronan (HA), is markedly elevated in lungs from patients and experimental models with PH. Herein, we identified HA synthase-2 (HAS2) in the pulmonary artery smooth muscle cell (PASMC) layer as a predominant locus of HA dysregulation. HA upregulation involves depletion of NUDT21, a master regulator of alternative polyadenylation, resulting in 3'UTR shortening and hyper-expression of HAS2. The ensuing increase of HAS2 and hyper-synthesis of HA promoted bioenergetic dysfunction of PASMC characterized by impaired mitochondrial oxidative capacity and a glycolytic shift. The resulting HA accumulation stimulated pro-remodeling phenotypes such as cell proliferation, migration, apoptosis-resistance, and stimulated pulmonary artery contractility. Transgenic mice, mimicking HAS2 hyper-synthesis in smooth muscle cells, developed spontaneous PH, whereas targeted deletion of HAS2 prevented experimental PH. Pharmacological blockade of HAS2 restored normal bioenergetics in PASMC, ameliorated cell remodeling phenotypes, and reversed experimental PH in vivo. In summary, our results uncover a novel mechanism of HA hyper-synthesis and downstream effects on pulmonary vascular cell metabolism and remodeling.