Tolerable glycometabolic stress boosts cancer cell resilience through altered N-glycosylation and Notch signaling activation.

Chronic metabolic stress paradoxically elicits pro-tumorigenic signals that facilitate cancer stem cell (CSC) development. Therefore, elucidating the metabolic sensing and signaling mechanisms governing cancer cell stemness can provide insights into ameliorating cancer relapse and therapeutic resistance. Here, we provide convincing evidence that chronic metabolic stress triggered by hyaluronan production augments CSC-like traits and chemoresistance by partially impairing nucleotide sugar metabolism, dolichol lipid-linked oligosaccharide (LLO) biosynthesis and N-glycan assembly. Notably, preconditioning with either low-dose tunicamycin or 2-deoxy-D-glucose, which partially interferes with LLO biosynthesis, reproduced the promoting effects of hyaluronan production on CSCs. Multi-omics revealed characteristic changes in N-glycan profiles and Notch signaling activation in cancer cells exposed to mild glycometabolic stress. Restoration of N-glycan assembly with glucosamine and mannose supplementation and Notch signaling blockade attenuated CSC-like properties and further enhanced the therapeutic efficacy of cisplatin. Therefore, our findings uncover a novel mechanism by which tolerable glycometabolic stress boosts cancer cell resilience through altered N-glycosylation and Notch signaling activation.

Trophoblast cell surface antigen-2 phosphorylation triggered by binding of galectin-3 drives metastasis through down-regulation of E-cadherin.

The expression of trophoblast cell surface antigen-2 (Trop-2) is enhanced in many tumor tissues and is correlated with increased malignancy and poor survival of patients with cancer. Previously, we demonstrated that the Ser-322 residue of Trop-2 is phosphorylated by protein kinase Cα (PKCα) and PKCδ. Here, we demonstrate that phosphomimetic Trop-2 expressing cells have markedly decreased E-cadherin mRNA and protein levels. Consistently, mRNA and protein of the E-cadherin-repressing transcription factors zinc finger E-Box binding homeobox 1 (ZEB1) were elevated, suggesting transcriptional regulation of E-cadherin expression. The binding of galectin-3 to Trop-2 enhanced the phosphorylation and subsequent cleavage of Trop-2, followed by intracellular signaling by the resultant C-terminal fragment. Binding of ß-catenin/transcription factor 4 (TCF4) along with the C-terminal fragment of Trop-2 to the ZEB1 promoter upregulated ZEB1 expression. Of note, siRNA-mediated knockdown of ß-catenin and TCF4 increased the expression of E-cadherin through ZEB1 downregulation. Knockdown of Trop-2 in MCF-7 cells and DU145 cells resulted in downregulation of ZEB1 and subsequent upregulation of E-cadherin. Furthermore, wild-type and phosphomimetic Trop-2 but not phosphorylation-blocked Trop-2 were detected in the liver and/or lung of some nude mice bearing primary tumors inoculated intraperitoneally or subcutaneously with wild-type or mutated Trop-2 expressing cells, suggesting that Trop-2 phosphorylation, plays an important role in tumor cell mobility in vivo, too. Together with our previous finding of Trop-2 dependent regulation of claudin-7, we suggest that the Trop-2-mediated cascade involves concurrent derangement of both tight and adherence junctions, which may drive metastasis of epithelial tumor cells.

Dysregulation of hexosamine biosynthetic pathway wiring metabolic signaling circuits in cancer.

Metabolite sensing, a fundamental biological process, plays a key role in metabolic signaling circuit rewiring. Hexosamine biosynthetic pathway (HBP) is a glucose metabolic pathway essential for the synthesis of uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), which senses key nutrients and integrally maintains cellular homeostasis. UDP-GlcNAc dynamically regulates protein N-glycosylation and O-linked-N-acetylglucosamine modification (O-GlcNAcylation). Dysregulated HBP flux leads to abnormal protein glycosylation, and contributes to cancer development and progression by affecting protein function and cellular signaling. Furthermore, O-GlcNAcylation regulates cellular signaling pathways, and its alteration is linked to various cancer characteristics. Additionally, recent findings have suggested a close association between HBP stimulation and cancer stemness; an elevated HBP flux promotes cancer cell conversion to cancer stem cells and enhances chemotherapy resistance via downstream signal activation. In this review, we highlight the prominent roles of HBP in metabolic signaling and summarize the recent advances in HBP and its downstream signaling, relevant to cancer.

The membrane-linked adaptor FRS2ß fashions a cytokine-rich inflammatory microenvironment that promotes breast cancer carcinogenesis.

Although it is held that proinflammatory changes precede the onset of breast cancer, the underlying mechanisms remain obscure. Here, we demonstrate that FRS2ß, an adaptor protein expressed in a small subset of epithelial cells, triggers the proinflammatory changes that induce stroma in premalignant mammary tissues and is responsible for the disease onset. FRS2ß deficiency in mouse mammary tumor virus (MMTV)-ErbB2 mice markedly attenuated tumorigenesis. Importantly, tumor cells derived from MMTV-ErbB2 mice failed to generate tumors when grafted in the FRS2ß-deficient premalignant tissues. We found that colocalization of FRS2ß and the NEMO subunit of the IκB kinase complex in early endosomes led to activation of nuclear factor-κB (NF-κB), a master regulator of inflammation. Moreover, inhibition of the activities of the NF-κB-induced cytokines, CXC chemokine ligand 12 and insulin-like growth factor 1, abrogated tumorigenesis. Human breast cancer tissues that express higher levels of FRS2ß contain more stroma. The elucidation of the FRS2ß-NF-κB axis uncovers a molecular link between the proinflammatory changes and the disease onset.

Hyaluronan: Metabolism and Function.

As a major polysaccharide component of the extracellular matrix, hyaluronan plays essential roles in the organization of tissue architecture and the regulation of cellular functions, such as cell proliferation and migration, through interactions with cell-surface receptors and binding molecules. Metabolic pathways for biosynthesis and degradation tightly control the turnover rate, concentration, and molecular size of hyaluronan in tissues. Despite the relatively simple chemical composition of this polysaccharide, its wide range of molecular weights mediate diverse functions that depend on molecular size and tissue concentration. Genetic engineering and pharmacological approaches have demonstrated close associations between hyaluronan metabolism and functions in many physiological and pathological events, including morphogenesis, wound healing, and inflammation. Moreover, emerging evidence has suggested that the accumulation of hyaluronan extracellular matrix and fragments due to the altered expression of hyaluronan synthases and hyaluronidases potentiates cancer development and progression by remodeling the tumor microenvironment. In addition to the well-known functions exerted by extracellular hyaluronan, recent metabolomic approaches have also revealed that its synthesis can regulate cellular functions via the reprogramming of cellular metabolism. This review highlights the current advances in knowledge on the biosynthesis and catabolism of hyaluronan and describes the diverse functions associated with hyaluronan metabolism.

Enhanced hexosamine metabolism drives metabolic and signaling networks involving hyaluronan production and O-GlcNAcylation to exacerbate breast cancer.

The hexosamine biosynthetic pathway (HBP) metabolically regulates dynamic cellular events by linking nutrient availability to numerous signaling networks. Significant alterations in the HBP are often associated with cancer pathogenesis. In this study, we investigated the molecular events underlying cancer pathogenesis associated with enhanced HBP flux. Multidimensional analysis of microarray datasets demonstrated up-regulation of genes encoding HBP enzymes in clinical breast cancers and revealed that co-expression of hyaluronan synthase 2 (HAS2) and glutamine:fructose-6-phosphate amidotransferase (GFAT), a rate-limiting enzyme of the HBP, was strongly correlated with a poor prognosis in advanced cancer patients. Consistently with the clinical data, comparative analyses of distinct breast cancer mouse models demonstrated enhancement of the HBP gene expression in primary carcinoma cells, with elevation of Has2 expression and hyaluronan production in aggressive breast cancer cells. The silencing of GFAT reduced CD44high/CD24low cancer stem cell (CSC)-like subpopulations, aldehyde dehydrogenase-positive cell populations, and mammosphere size, which were further diminished by gene targeting of Has2. Has2 gene disruption reduced the in vivo growth of aggressive cancer cells and attenuated pro-tumorigenic Akt/GSK3ß/ß-catenin signaling and cisplatin resistance. Overall protein O-GlcNAcylation was also elevated in association with HBP enhancement in aggressive cancer cells, and the modification exhibited overlapping but distinct roles from the hyaluronan signal in the regulation of CSC-like features. The current data therefore demonstrate that enhanced hexosamine metabolism drives pro-tumorigenic signaling pathways involving hyaluronan and O-GlcNAcylation in aggressive breast cancer.

Implications of altered O-glycosylation in tumour immune evasion.

Aberrant glycosylation on tumour cells has been implicated in tumour immune modulation. A recent article published in The Journal of Biochemistry (Sutoh Yoneyama et al., A mechanism for evasion of CTL immunity by altered O-glycosylation of HLA class I, J. Biochem. 2017;161:479-492) showed that bladder cancer cells evaded cytotoxic T lymphocyte-mediated antitumour immunity by a novel mechanism involving the loss of Core 2 structures on human leukocyte antigen Class I O-glycans and subsequent impairment of galectin-glycan lattice formation. The immunosuppressive action of O-glycans on natural killer cell-mediated tumour immunity is also considered an immune evasion system. Furthermore, sialylated O-glycans have been proposed to play a central role in tumour immune escape by modulating the production of immunoregulatory cytokines and growth factors through interactions with sialic acid-binding immunoglobulin-like lectins. Therefore, a better understanding of how alterations in O-glycosylation influence tumour immune evasion will enable the development of novel and more effective therapeutic options for cancer treatment.

Hyaluronan Production Regulates Metabolic and Cancer Stem-like Properties of Breast Cancer Cells via Hexosamine Biosynthetic Pathway-coupled HIF-1 Signaling.

Cancer stem cells (CSCs) represent a small subpopulation of self-renewing oncogenic cells. As in many other stem cells, metabolic reprogramming has been implicated to be a key characteristic of CSCs. However, little is known about how the metabolic features of cancer cells are controlled to orchestrate their CSC-like properties. We recently demonstrated that hyaluronan (HA) overproduction allowed plastic cancer cells to revert to stem cell states. Here, we adopted stable isotope-assisted tracing and mass spectrometry profiling to elucidate the metabolic features of HA-overproducing breast cancer cells. These integrated approaches disclosed an acceleration of metabolic flux in the hexosamine biosynthetic pathway (HBP). A metabolic shift toward glycolysis was also evident by quantitative targeted metabolomics, which was validated by the expression profiles of key glycolytic enzymes. Forced expression of glutamine:fructose-6-phosphate amidotransferase 1 (GFAT1), an HBP rate-limiting enzyme, resembled the results of HA overproduction with regard to HIF-1α accumulation and glycolytic program, whereas GFAT1 inhibition significantly decreased HIF-1α protein level in HA-overproducing cancer cells. Moreover, inhibition of the HBP-HIF-1 axis abrogated HA-driven glycolytic enhancement and reduced the CSC-like subpopulation. Taken together, our results provide compelling evidence that HA production regulates the metabolic and CSC-like properties of breast cancer cells via HBP-coupled HIF-1 signaling.

Hyaluronan: A modulator of the tumor microenvironment.

Tumors are cellular masses formed through dynamic interactions between tumor cells and a mixed population of stromal cells. Crosstalk between oncogenic and adjacent stromal cells contributes to the formation of a "tumor microenvironment" influencing the tumor cell behaviors of proliferation, invasion, and metastatic spread throughout cancer progression. The composition and structure of the tumor microenvironment vary among different types of tumors and are extensively remodeled in close association with tumor advancement. The tumor microenvironment is composed not only of cellular compartments, such as endothelial cells, fibroblasts, inflammatory cells, and immune cells, but also of bioactive substances, including growth factors and the extracellular matrix. Hyaluronan (HA) is a major component of the extracellular matrix, and the degree of HA accumulation is strongly correlated with a poor prognosis in advanced cancer patients. Emerging evidence has suggested that HA creates a specific microenvironment that is favorable for tumor angiogenesis, invasion, and metastasis. This review highlights the prominent roles of HA as a modulator of the tumor microenvironment and addresses the recent advances regarding HA function in cancer stem cell niches.

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

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

Excessive hyaluronan production promotes acquisition of cancer stem cell signatures through the coordinated regulation of Twist and the transforming growth factor ß (TGF-ß)-Snail signaling axis.

The cancer stem cell (CSC) model suggests that a small subpopulation of cancer cells possesses the ability to self-renew and give rise to malignant progeny that drive cancer progression. Recent reports have also proposed the existence of certain extra- or intracellular signals that allow cancer progenitors to dynamically revert to a stem cell state. However, the mechanisms underlying cancer cell plasticity and CSC expansion are not entirely clear. Our previous studies using a hyaluronan synthase 2 (Has2) transgenic mouse model demonstrated that hyaluronan overproduction caused rapid development of aggressive breast carcinoma at a high incidence. Thus, we hypothesize that hyaluronan overproduction may accelerate cancer progression by expanding CSC subpopulations during cancer development. Primary cancer cells were established from mammary tumors developed in the transgenic mice and subjected to the Hoechst 33342 dye exclusion assay to sort side population (SP) from non-side population (non-SP) cells. Flow cytometric analysis demonstrated the enrichment of CD44(high)/CD24(low) CSC-like cells in the SP fraction of hyaluronan-overproducing cancer cells. This subpopulation exhibited several characteristics that were similar to CSCs, including cancer-initiating and mammosphere-forming abilities. Excess hyaluronan production drove the epithelial-to-mesenchymal transition process defined as the loss of epithelial phenotypes, up-regulation of transforming growth factor ß (TGF-ß), and induction of the epithelial-to-mesenchymal transition-related transcriptional factors Snail and Twist. Inhibition of TGF-ß-Snail signaling or silencing of Twist expression abrogated the entrance into a stem cell state. Taken together, our findings suggest that hyaluronan overproduction allows plastic cancer cell populations to revert to stem cell states via Twist and the TGF-ß-Snail signaling axis.

Tumor-associated macrophages as major players in the tumor microenvironment.

During tumor progression, circulating monocytes and macrophages are actively recruited into tumors where they alter the tumor microenvironment to accelerate tumor progression. Macrophages shift their functional phenotypes in response to various microenvironmental signals generated from tumor and stromal cells. Based on their function, macrophages are divided broadly into two categories: classical M1 and alternative M2 macrophages. The M1 macrophage is involved in the inflammatory response, pathogen clearance, and antitumor immunity. In contrast, the M2 macrophage influences an anti-inflammatory response, wound healing, and pro-tumorigenic properties. Tumor-associated macrophages (TAMs) closely resemble the M2-polarized macrophages and are critical modulators of the tumor microenvironment. Clinicopathological studies have suggested that TAM accumulation in tumors correlates with a poor clinical outcome. Consistent with that evidence, experimental and animal studies have supported the notion that TAMs can provide a favorable microenvironment to promote tumor development and progression. In this review article, we present an overview of mechanisms responsible for TAM recruitment and highlight the roles of TAMs in the regulation of tumor angiogenesis, invasion, metastasis, immunosuppression, and chemotherapeutic resistance. Finally, we discuss TAM-targeting therapy as a promising novel strategy for an indirect cancer therapy.

Visible, safe and certain endotracheal intubation using endoscope system and inhalation anesthesia for rats.

Anesthesia strongly influences laboratory animals, and it can also greatly affect the experimental data. Rats rank only second to mice in the number used in research fields, such as organ transplantation, regenerative medicine and imaging. Therefore, appropriate and effective anesthesia, including the protocol of the endotracheal intubation and inhalation anesthesia, is crucial. Hence, we evaluated these methods in this study. Twelve Wistar rats were intraperitoneally injected with M/M/B: 0.3/4/5, comprising of medetomidine, midazolam and butorphanol at a dose of 0.3 mg/kg + 4.0 mg/kg + 5.0 mg/kg body weight/rat, respectively. An endotracheal tube was then intubated into the trachea. After intubation, the rats were connected to the inhalation anesthesia circuit using isoflurane, and vital signs were measured until 30 min after connection. All intubations were successfully finished within 1 min, and the values of the vital signs were normal and stable. In addition, histopathological observation of the trachea and lungs showed no trauma. These results suggest that this visible endotracheal intubation method is simple, reliable, safe and favorable with regard to the rats' welfare.

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

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

New visible endotracheal intubation method using the endoscope system for mice inhalational anesthesia.

Appropriate and effective anesthesia is critical, because it has a strong influence on laboratory animals, and its affect greatly impacts the experimental data. Inhalational anesthesia by endotracheal intubation is currently prevailing in general anesthesia and is prefered over injection anesthesia, especially for large laboratory animals, because it is a safe and easy control agent. However, it is not common for small laboratory animals, because of the high degree of technical skills required. We assessed the capability of use for mice of the endotracheal intubation by using the endoscope system "TESALA AE-C1" and inhalational anesthesia using a ventilator. Endotracheal intubation was successfully performed on all 10 C57BL/6 mice injected with M/M/B: 0.3/4/5 comprised of medetomidine, midazoram and butorphanol, at a dose of 0.3 mg/kg + 4.0 mg/kg + 5.0 mg/kg body weight/mouse, respectively. After the intubated mice were connected with the inhalational anesthesia circuit and the ventilator, vital signs were measured until 15 min after the connection. The data with M/M/B: 0.3/4/5 showed stable and normal values, which indicated that this new endotracheal intubation method was simple, reliable and safe, which mean that this anesthesia is favorable in regard to the animal's welfare.

Effect of a cholesterol-rich lipid environment on the enzymatic activity of reconstituted hyaluronan synthase.

Hyaluronan synthase (HAS) is a unique membrane-associated glycosyltransferase and its activity is lipid dependent. The dependence however is not well understood, especially in vertebrate systems. Here we investigated the functional association of hyaluronan synthesis in a cholesterol-rich membrane-environment. The culture of human dermal fibroblasts in lipoprotein-depleted medium attenuated the synthesis of hyaluronan. The sequestration of cellular cholesterol by methyl-ß-cyclodextrin also decreased the hyaluronan production of fibroblasts, as well as the HAS activity. To directly evaluate the effects of cholesterol on HAS activity, a recombinant human HAS2 protein with a histidine-tag was expressed as a membrane protein by using a baculovirus system, then successfully solubilized, and isolated by affinity chromatography. When the recombinant HAS2 proteins were reconstituted into liposomes composed of both saturated phosphatidylcholine and cholesterol, this provided a higher enzyme activity as compared with the liposomes formed by phosphatidylcholine alone. Cholesterol regulates HAS2 activity in a biphasic manner, depending on the molar ratio of phosphatidylcholine to cholesterol. Furthermore, the activation profiles of different lipid compositions were determined in the presence or absence of cholesterol. Cholesterol had the opposite effect on the HAS2 activity in liposomes composed of phosphatidylethanolamine or phosphatidylserine. Taken together, the present data suggests a clear functional association between HAS activity and cholesterol-dependent alterations in the physical and chemical properties of cell membranes.

ASC-associated inflammation promotes cecal tumorigenesis in aryl hydrocarbon receptor-deficient mice.

The aryl hydrocarbon receptor (AhR) plays a suppressive role in cecal carcinogenesis by CUL4B/AhR-mediated ubiquitylation and degradation of ß-catenin, which is activated by xenobiotics and natural ligands. AhR-deficient (AhR(-)(/-)) mice develop cecal tumors with severe inflammation. To elucidate whether the tumors develop autonomously in AhR(-/-) mice due to impaired ß-catenin degradation or in association with accelerated inflammation, we performed two kinds of experiments using germ-free (GF) AhR(-/-) mice and compound mutant mice lacking genes for AhR and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), which plays an essential role in caspase-1 activation in inflammasomes. Both GF AhR(-/-) and AhR(-/-)•ASC(-/-) mice showed considerably reduced tumor development compared with that in AhR(-/-) mice albeit in a 'cancer-prone' state with aberrant ß-catenin accumulation. Blocking of the interleukin (IL)-1ß signaling pathway by treatment with a caspase-1 inhibitor, YVAD, reduced cecal tumorigenesis in AhR(-/-) mice. Signal transducers and activators of transcription 3 (STAT3) activation was detected in the cecal epithelium of the AhR(-/-) mice due to enhanced IL-6 production. An inhibitor of the STAT3 signaling pathway, AG490 suppressed the tumor formation. ASC-mediated inflammation was also found to play a critical role in tumor development in Apc(Min/+) mice, a mouse model of familial adenomatous polyposis. Collectively, these results revealed an important role of the bacteria-triggered or ASC-mediated inflammation signaling pathway in the intestinal tumorigenesis of mice and suggest a possible chemical therapeutic intervention, including AhR ligands and inhibitors of the inflammation pathway.

Crucial role of hyaluronan in neointimal formation after vascular injury.

BACKGROUND: Hyaluronan (HA) is a primary component of the extracellular matrix of cells, and it is involved in the pathogenesis of atherosclerosis. The purpose of this study was to investigate the role of HA in neointimal formation after vascular injury and determine its tissue-specific role in vascular smooth muscle cells (VSMCs) by using a cre-lox conditional transgenic (cTg) strategy. METHODS AND RESULTS: HA was found to be expressed in neointimal lesions in humans with atherosclerosis and after wire-mediated vascular injury in mice. Inhibition of HA synthesis using 4-methylumbelliferone markedly inhibited neointimal formation after injury. In vitro experiments revealed that low-molecular-weight HA (LMW-HA) induced VSMC activation, including migration, proliferation, and production of inflammatory cytokines, and reactive oxygen species (ROS). The migration and proliferation of VSMCs were mediated by the CD44/RhoA and CD44/ERK1/2 pathways, respectively. Because HA synthase 2 (HAS2) is predominantly expressed in injured arteries, we generated cTg mice that overexpress the murine HAS2 gene specifically in VSMCs (cHAS2/CreSM22α mice) and showed that HA overexpression markedly enhanced neointimal formation after cuff-mediated vascular injury. Further, HA-overexpressing VSMCs isolated from cHAS2/CreSM22α mice showed augmented migration, proliferation, and production of inflammatory cytokines and ROS. CONCLUSION: VSMC-derived HA promotes neointimal formation after vascular injury, and HA may be a potential therapeutic target for cardiovascular disease.

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

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

Restriction of mast cell proliferation through hyaluronan synthesis by co-cultured fibroblasts.

Appropriate culture models for tissue mast cells are required to determine how they are involved in regulation of local immune responses. We previously established a culture model for cutaneous mast cells, in which bone marrow-derived immature mast cells were co-cultured with Swiss 3T3 fibroblasts in the presence of stem cell factor. In this study, we focused on the roles of hyaluronan, which is produced by the feeder fibroblasts and forms the extracellular matrix during the co-culture period. Hyaluronan synthesis was found to be mediated by hyaluronan synthase 2 (HAS2) expressed in Swiss 3T3 cells. A decreases in the amount of hyaluronan, which was achieved by retroviral expression of short hairpin RNA for Has2 or by addition of hyaluronidase, significantly enhanced the proliferation of the cultured mast cells without any obvious effects on their maturation. Although we previously demonstrated that CD44 is required for proliferation of cutaneous mast cells, the deficiency of hyaluronan did not affect the proliferation of the cultured mast cells that lack CD44. These findings suggest that the extracellular matrix containing hyaluronan may have a potential to restrict proliferation of cutaneous mast cells in a CD44-independent manner.