Expression Of Hyaluronan Synthase Research Articles

Developmental pathways and endothelial to mesenchymal transition in canine myxomatous mitral valve disease

Epithelial to mesenchymal transition (EMT) and the cardiovascular equivalent, endothelial to mesenchymal transition (EndoMT), contribute to a range of chronic degenerative diseases and cancer metastasis. Chronic valvulopathies exhibit some features of EndoMT and activation of developmental signalling pathways, such as osteogenesis and chondrogenesis, expression of cell differentiation markers, basement membrane damage and endothelial transformation. The aim of the present study was to investigate the potential role of developmental mechanisms in canine myxomatous mitral valve disease (MMVD) using a combination of transcriptomic array technology, RT-PCR and immunohistochemistry.There was significant differential expression for genes typically associated with valvulogenesis and EndoMT, including markers of inflammation (IL6, IL18 and TLR4), basement membrane disarray (NID1, LAMA2 and CTSS), mesenchymal and endothelial cell differentiation (MYH11 and TAGLN) and EndoMT (ACTA2, SNAI1, CTNNB1, HAS2, CDH5, and NOTCH1), with fold changes from +15.35 (ACTA2) to −5.52 (LAMA2). These changes in gene expression were confirmed using RT-PCR, except for HAS2. In silico analysis identified important gene networks and canonical pathways in MMVD that have associations with development and organogenesis, including inflammation, valve morphogenesis and EMT, as well as components of the basement membrane and extra-cellular matrix. Immunohistochemistry identified changes in the expression of hyaluronic acid synthase (Has2), Snai1, α-smooth muscle actin (α-SMA) and VE-cadherin (CDH5), and co-expression of Has2 with α-SMA. These research findings strongly suggest involvement of developmental signalling pathways and mechanisms, including EndoMT, in the pathogenesis of canine MMVD.

Abstract 4949: HAS2 is a critical effector for AGL mediated regulation of tumor growth

Abstract In bladder cancer, reduced levels of Amylo-alpha-1-6-glucosidase-4-alpha-glucanotransferase (AGL), an enzyme involved in glycogenolysis and mutated in glycogen storage disease type III, enhances proliferation in vitro and tumor growth in vivo. To identify how reduced levels of AGL promote bladder cancer growth, we gene expression profiled two bladder cancer cell lines with and without siRNA mediated AGL depletion. This identified that hyaluronic acid synthase 2 (HAS2), an enzyme responsible for hyaluronic acid (HA) synthesis, is upregulated with AGL depletion. We validated this finding in several additional bladder cancer cell lines and also found that HA levels were 2-fold higher bladder cancer cells with low AGL compared to control. Interestingly, siRNA induced knockdown of HAS2 preferentially reduced monolayer, anchorage independent and xenograft growth in bladder cancer cells with low AGL. 4-Methylumbelliferone (4-MU), an inhibitor of HA synthesis, had similar effects. Analysis of human bladder cancer tissues showed that AGL and HAS2 mRNA expression are negatively correlated in 5/8 patient datasets (N = 725). Bladder cancer patients with high HAS2 and low AGL expression had worse survival than patients with the reciprocal relationship between these two genes suggesting that HAS2 is a driver of bladder tumor growth with AGL loss establish the HAS2/HA axis as a major driver and target of therapy in bladder tumors with low AGL. Citation Format: Sunny Guin, Yuanbin Ru, Carolyn R. Lew, Neeraj Agarwal, Charles Owens, Dan Theodorescu. HAS2 is a critical effector for AGL mediated regulation of tumor growth. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4949. doi:10.1158/1538-7445.AM2015-4949

Transforming growth factor-β1 induces EMT by the transactivation of epidermal growth factor signaling through HA/CD44 in lung and breast cancer cells.

Epithelial-mesenchymal transition (EMT), a process closely related to tumor development, is regulated by a variety of signaling pathways and growth factors, such as transforming growth factor-β1 (TGF-β1) and epidermal growth factor (EGF). Hyaluronan (HA) has been shown to induce EMT through either TGF-β1 or EGF signaling and to be a regulator of the crosstalk between these two pathways in fibroblasts. In this study, in order to clarify whether HA has the same effect in tumor cells, we utilized the lung cancer cell line, A549, and the breast cancer cell line, MCF-7, and found that the effects of stimulation with TGF-β1 were more potent than those of EGF in regulating the expression of EMT-associated proteins and in enhancing cell migration and invasion. In addition, we observed that TGF-β1 activated EGF receptor (EGFR) and its downstream AKT and extracellular signal-regulated kinase (ERK) pathways. Furthermore, we found that TGF-β1 upregulated the expression of hyaluronan synthases (HAS1, HAS2 and HAS3) and promoted the expression of CD44, a cell surface receptor for HA, which interacts with EGFR, resulting in the activation of the downstream AKT and ERK pathways. Conversely, treatment with 4-methylumbelliferone (4-MU; an inhibitor of HAS) prior to stimulation with TGF-β1, inhibited the expression of CD44 and EGFR, abolished the interaction between CD44 and EGFR. Furthermore, the use of shRNA targeting CD44 impaired the expression of EGFR, deactivated the AKT and ERK pathways, reversed EMT and decreased the migration and invasion ability of cells. In conclusion, our data demonstrate that TGF-β1 induces EMT by the transactivation of EGF signaling through HA/CD44 in lung and breast cancer cells.

Abstract P4-04-25: Naked mole rat hyaluronan synthase 2 displays similar effects as human hyaluronan synthase 2 and promotes tumor growth in a mouse xenograft model

Abstract Accumulation of hyaluronan (HA) is correlated with poor prognosis in many human cancer including breast cancer and pancreatic cancer. Hyaluronan synthase 2 (HAS2) is one of the three HA synthase genes (HAS1-3) found in mammals. Up-regulation of the HAS2 gene has been observed in breast cancer patients; and HAS2 expression correlates with the incidence of metastasis and lower rate of overall patient survival (Okuda et al 2012). Interestingly, a recent study suggested that the naked mole rat (nmr) HAS2 enzyme may function differently than the HAS2 proteins from mice and humans, and its expression had a tumor suppressor effect (Tian et al 2013). The nmrHAS2 protein sequence was distinct from other mammals in the cytoplasmic loop where the active site resides, which led to the hypothesis that nmrHAS2 may contribute to the production of extremely high molecular weight HA species. Furthermore, nmrHAS2 is required for the resistance of nmr skin fibroblasts to malignant transformation. In this study, we examined whether the nmrHAS2 gene can also render human cells resistant to tumor progression. We have previously observed that HA level in the pancreatic cancer model AsPC1 can be modulated by the over-expression of the human HAS3 protein (Osgood et al 2014). To compare and contrast the effect of nmrHAS2 and huHAS2 on HA production and tumor growth, nmrHAS2 and huHAS2 AsPC1 models were engineered using a recombinant lentivirus encoding the nmrHAS2 and huHAS2 genes respectively. Over-expression of nmrHAS2 and huHAS2 both significantly increased the HA level in AsPC1 cells. Both nmrHAS2 and huHAS2 promoted the growth of AsPC1 in an intramuscular xenograft tumor model. Ex vivo analysis of tumor xenografts showed that nmrHAS2 and huHAS2 AsPC1 tumors contain elevated levels of HA, and the size range of the HA in the nmrHAS2 tumors is similar to that in the huHAS2 AsPC1 tumors. Therefore, nmrHAS2 functions similarly as huHAS2 gene and promotes tumor growth in the human pancreatic tumor model AsPC1.

20-O-β-D-glucopyranosyl-20(S)-protopanaxadiol, a metabolite of ginsenoside Rb1, enhances the production of hyaluronic acid through the activation of ERK and Akt mediated by Src tyrosin kinase in human keratinocytes.

The aim of the present study was to determine the mechanisms through which 20-O-β-D-glucopyranosyl-20(S)-protopanaxadiol (20GPPD) promotes the production of hyaluronic acid (HA) in human keratinocytes. 20GPPD is the primary bioactive metabolite of Rb1, a major ginsenoside found in ginseng (Panax ginseng). We sought to elucidate the underlying mechanisms behind the 20GPPD-induced production of HA. We found that 20GPPD induced an increase in HA production by elevating hyaluronan synthase 2 (HAS2) expression in human keratinocytes. The phosphorylation of extracellular signal-regulated kinase (ERK) and Akt was also enhanced by 20GPPD in a dose-dependent manner. The pharmacological inhibition of ERK (using U0126) or Akt (using LY294002) suppressed the 20GPPD-induced expression of HAS2, whereas treatment with an epidermal growth factor receptor (EGFR) inhibitor (AG1478) or an intracellular Ca2+ chelator (BAPTA/AM) did not exert any observable effects. The increased Src phosphorylation was also confirmed following treatment with 20GPPD in the human keratinocytes. Following pre-treatment with the Src inhibitor, PP2, both HA production and HAS2 expression were attenuated. Furthermore, the 20GPPD-enhanced ERK and Akt signaling decreased following treatment with PP2. Taken together, our results suggest that Src kinase plays a critical role in the 20GPPD-induced production of HA by acting as an upstream modulator of ERK and Akt activity in human keratinocytes.

Expression of focal adhesion kinase in mouse cumulus-oocyte complexes, and effect of phosphorylation at Tyr397 on cumulus expansion.

We investigated the expression of focal adhesion kinase (FAK) in mouse cumulus-oocyte complexes (COCs), as well as the role of FAK phosphorylation at Tyr397 during oocyte maturation. The effect of inhibiting FAK phosphorylation at Tyr397 during in vitro maturation (IVM) on subsequent fertilization and preimplantation embryo development was also examined. Western blotting analyses revealed that total and Tyr397-phosphorylated FAK were expressed in vivo in both cumulus cells and oocytes. Immunocytochemical studies localized this kinase throughout the cytoplasm of cumulus cells and oocytes; in particular, Tyr397-phosphorylated FAK tended to accumulate in regions where cumulus cells contact each other. Interestingly, the in vivo level of Tyr397 phosphorylation in cumulus cells was significantly lower after compared to before cumulus expansion. Addition of FAK inhibitor 14, which specifically blocks phosphorylation at Tyr397, stimulated oocyte meiotic maturation and cumulus expansion during IVM in the absence of follicle-stimulating hormone (FSH). Reverse-transcriptase PCR showed that the mRNA expression of hyaluronan synthase 2 (Has2), a marker of cumulus expansion, was significantly induced in cumulus cells. Subsequent in vitro fertilization and culture showed that more oocytes developed to the blastocyst stage when they were treated with FAK inhibitor 14 during IVM, although the blastocyst total cell number was lower than in oocytes stimulated with FSH. These results indicate that FAK is involved in the maturation of COCs; specifically, phosphorylation at Tyr397 may regulate cumulus expansion via the expression of Has2 mRNA in cumulus cells, which could affect the developmental competence of oocytes.

Soluble CD40L promotes proliferation of orbital fibroblasts and expression of hyaluronan synthase in patients with thyroid associated ophthalmopathy

Objective To investigate the effects of soluble CD40L(sCD40L)on proliferation of orbital fibroblasts(OFs)and the expression of three types of hyaluronan synthase(HAS)in vitro,so as to explore the role of sCD40 Lin the pathogenesis of thyroid-associated ophthalmopathy(TAO).Methods OFs obtained from 5patients with TAO and 3normal controls were primarily culutred.The effect of different concentrations of sCD40L(6.25,12.5,25,50,100 and 200ng/mL)on proliferation of OFs of different sources were examined by MTS after 48 hexposure.OFs were also cultured with different concentrations of sCD40L(12.5,25,50 and 100ng/mL)for 3,6,12 and 24h,and then the expression levels of HAS1-3mRNA were determined by real-time RT-PCR.Results Treatment with sCD40 Lat concentrations higher than 25ng/mL for 48 h obviously promoted the proliferation of OFs in patients with TAO(P0.05).In contrast,treatment with sCD40 Lonly at concentrations higher than 50ng/mL for 48 hcould promote proliferation of OFs from normal control,and the effect was comparatively weak.HAS3 mRNA expression of OFs in TAO patients was increased after exposed to sCD40L(P0.05),and the increase was in a concentration-and time-dependent manner.Conclusion sCD40 Lcan promote the proliferation of OFs and expression of HAS3 mRNA in patients with TAO,which implies that sCD40 Lplays an important role in the pathogenesis of TAO.

Regulation of synthesis and roles of hyaluronan in peritoneal dialysis.

Hyaluronan (HA) is a ubiquitous extracellular matrix glycosaminoglycan composed of repeated disaccharide units of alternating D-glucuronic acid and D-N-acetylglucosamine residues linked via alternating β-1,4 and β-1,3 glycosidic bonds. HA is synthesized in humans by HA synthase (HAS) enzymes 1, 2, and 3, which are encoded by the corresponding HAS genes. Previous in vitro studies have shown characteristic changes in HAS expression and increased HA synthesis in response to wounding and proinflammatory cytokines in human peritoneal mesothelial cells. In addition, in vivo models and human peritoneal biopsy samples have provided evidence of changes in HA metabolism in the fibrosis that at present accompanies peritoneal dialysis treatment. This review discusses these published observations and how they might contribute to improvement in peritoneal dialysis.

Abstract B84: Activation of the STAT3 pathway leads to a hyaluronan-rich microenvironment that promotes breast cancer growth and therapeutic resistance

Abstract As breast tumors form, complex interactions between the tumor cells and the surrounding stroma create a microenvironment that promotes tumor growth, resistance to therapy and recurrence. Our recent studies have focused on hyaluronan (HA), a component of the extracellular matrix that is present in the normal breast stroma but is altered at the levels of expression and fragmentation during tumor growth and progression. We have found that activation of fibroblast growth factor receptor (FGFR), which has been implicated in both triple negative and endocrine resistant breast cancers, leads to increased HA in the microenvironment. Furthermore, we have found that blocking HA synthesis decreases proliferation, migration and therapeutic resistance. Analysis of the mechanisms responsible for regulating HA synthesis revealed that FGFR-mediated HA accumulation requires activation of the signal transducer and activator of transcription 3 (STAT3) pathway, which regulates expression of hyaluronan synthase 2 (HAS2) and subsequent HA synthesis. Using a novel in vivo model of FGFR-dependent tumor growth, we demonstrate that STAT3 inhibition decreases both FGFR-driven tumor growth and HA levels within the tumor. Finally, our results suggest that combinatorial therapies inhibiting both FGFR activity and HA synthesis are more effective than targeting either pathway alone and may be a relevant therapeutic approach for breast cancers associated with high levels of FGFR activity. Increased synthesis and fragmentation of HA are typically associated with inflammatory responses. Thus, we have expanded our studies to evaluate HA in the context of tumor-associated inflammation. Interestingly, we have found that macrophage-derived inflammatory cytokines also contribute to altered HA metabolism, linking inflammation to HA-regulated tumorigenesis. These studies suggest that tumor cells and innate immune cells cooperate to enhance the pro-tumorigenic properties of HA in the microenvironment of the developing tumor. Furthermore, we have found that HA acts through specific receptors on both tumor cells and macrophages to promote tumorigenesis, suggesting multiple mechanisms through which HA is capable of contributing to tumor formation. In conclusion, we have found that tumor cells and macrophages cooperate to induce tumor-promoting changes in the HA-containing microenvironment. Ongoing studies are focused on developing strategies that target both the tumor cells and the HA-containing microenvironment, which will ultimately lead to enhanced therapeutic efficacy for breast cancer patients. Citation Format: Laura R. Bohrer, Pavlina Chuntova, James B. McCarthy, Kathryn L. Schwertfeger. Activation of the STAT3 pathway leads to a hyaluronan-rich microenvironment that promotes breast cancer growth and therapeutic resistance. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B84. doi:10.1158/1538-7445.CHTME14-B84

Hyaluronan synthase-2 upregulation protects smpd3-deficient fibroblasts against cell death induced by nutrient deprivation, but not against apoptosis evoked by oxidized LDL

The neutral type 2 sphingomyelinase (nSMase2) hydrolyzes sphingomyelin and generates ceramide, a major bioactive sphingolipid mediator, involved in growth arrest and apoptosis. The role of nSMase2 in apoptosis is debated, and apparently contradictory results have been observed on fibroblasts isolated from nSMase2-deficient fragilitas ossium (homozygous fro/fro) mice. These mice exhibit a severe neonatal dysplasia, a lack of long bone mineralization and delayed apoptosis patterns of hypertrophic chondrocytes in the growth plate. We hypothesized that apoptosis induced by nutrient deprivation, which mimics the environmental modifications of the growth plate, requires nSMase2 activation. In this study, we have compared the resistance of fro/fro fibroblasts to different death inducers (oxidized LDL, hydrogen peroxide and nutrient starvation). The data show that nSMase2-deficient fro/fro cells resist to apoptosis evoked by nutrient starvation (fetal calf serum/glucose/pyruvate-free DMEM), whereas wt fibroblasts die after 48h incubation in this medium. In contrast, oxidized LDL and hydrogen peroxide are similarly toxic to fro/fro and wt fibroblasts, indicating that nSMase2 is not involved in the mechanism of toxicity evoked by these agents. Interestingly, wt fibroblasts treated with the SMase inhibitor GW4869 were more resistant to starvation-induced apoptosis.The resistance of fro/fro cells to starvation-induced apoptosis is associated with an increased expression of hyaluronan synthase 2 (HAS2) mRNAs and protein, which is inhibited by ceramide. In wt fibroblasts, this HAS2 rise and its protective effect did not occur, but exogenously added HA exhibited a protective effect against starvation-induced apoptosis.The protective mechanism of HAS2 involves an increased expression of the heat-shock protein Hsp72, a chaperone with antiapoptotic activity. Taken together, these results highlight the role of nSMase2 in apoptosis evoked by nutrient starvation that could contribute to the delayed apoptosis of hypertrophic chondrocytes in the growth plate, and emphasize the antiapoptotic properties of HAS2.

Homeostasis of Hyaluronic Acid in Normal and Scarred Vocal Folds

Vocal fold scarring is one of the most challenging laryngeal disorders to treat. Hyaluronic acid (HA) is the main component of lamina propria, and it plays an important role in proper vocal fold vibration and is also thought to be important in fetal wound healing without scarring. Although several animal models of vocal fold scarring have been reported, little is known about the way in which HA is maintained in vocal folds. The purpose of this study was to clarify the homeostasis of HA by examining the expression of hyaluronan synthase (Has) and hyaluronidase (Hyal), which produce and digest HA, respectively. Experimental prospective animal study. Vocal fold stripping was performed on 38 Sprague-Dawley rats. Vocal fold tissue was collected at five time points (3days-2months). Expression of HA was examined by immunohistochemistry, and messenger RNA (mRNA) expression of Has and Hyal was examined by real-time polymerase chain reaction and in-situ hybridization. In scarred vocal folds, expression of Has1 and Has2 increased at day 3 together with expression of HA and returned to normal at 2weeks. At 2months, Has3 and Hyal3 mRNA showed higher expressions than normal. Expression patterns of Has and Hyal genes differed between normal, acute-scarred, and chronic-scarred vocal folds, indicating the distinct roles of each enzyme in maintaining HA. Continuous upregulation of Has genes in the acute phase may be necessary to achieve scarless healing of vocal folds.

A Role for Versican in the Development of Leiomyosarcoma

Leiomyosarcoma (LMS) is a mesenchymal cancer that occurs throughout the body. Although LMS is easily recognized histopathologically, the cause of the disease remains unknown. Versican, an extracellular matrix proteoglycan, increases in LMS. Microarray analyses of 80 LMSs and 24 leiomyomas showed a significant elevated expression of versican in human LMS versus benign leiomyomas. To explore the importance of versican in this smooth muscle cell tumor, we used versican-directed siRNA to knock down versican expression in a LMS human cell line, SK-LMS-1. Decreased versican expression was accompanied by slower rates of LMS cell proliferation and migration, increased adhesion, and decreased accumulation of the extracellular matrix macromolecule hyaluronan. Addition of purified versican to cells expressing versican siRNA restored cell proliferation to the level of LMS controls, increased the pericellular coat and the retention of hyaluronan, and decreased cell adhesion in a dose-dependent manner. The presence of versican was not only synergistic with hyaluronan in increasing cell proliferation, but the depletion of versican decreased hyaluronan synthase expression and decreased the retention of hyaluronan. When LMS cells stably expressing versican siRNA were injected into nude mice, the resulting tumors displayed significantly less versican and hyaluronan staining, had lower volumes, and had reduced levels of mitosis as compared with controls. Collectively, these results suggest a role for using versican as a point of control in the management and treatment of LMS.

4-Methylumbelliferone inhibits ovarian cancer growth by suppressing thymidine phosphorylase expression.

Background4-Methylumbelliferone (4-MU), a hyaluronan (HA) synthesis inhibitor, has antitumor activity in cancer cells. However, few studies have focused on its effects on ovarian cancer. The aim of this study was to investigate the effects of 4-MU on ovarian cancer and to elucidate its mechanism of action.MethodsThe HRA human ovarian serous adenocarcinoma cell line was used in this study. The effects of 4-MU on cell proliferation, migration, and invasion were determined by using in vitro assays as well as an in vivo rat peritoneal carcinomatosis model. The expression of HA synthase (HAS), CD44 HA receptor, vascular endothelial growth factor (VEGF), and thymidine phosphorylase (TP) mRNA in HRA cells was analyzed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR).Results4-MU administration inhibited the growth of peritoneal tumors and significantly prolonged survival. In vitro experiments showed that 4-MU inhibited HRA cell proliferation in a dose-dependent manner, while it did not affect HRA cell invasion and migration. 4-MU significantly decreased TP mRNA expression in HRA cells. On the other hand, since HAS2, CD44, and VEGF endogenous mRNA expression levels were very low in HRA cells, it was impossible to evaluate the effect of 4-MU treatment.ConclusionsThese results suggest that 4-MU exerts its antitumor effect on ovarian cancer through suppressing TP expression.

Abstract 3023: The characterisation of hyaluronan-related enzymes in breast cancer cell subpopulations

Abstract Background:Hyaluronan (HA) has been associated with various facets of tumor biology where it provides structural integrity, maintains hydration homeostasis, and in conjunction with its metabolic receptor, CD44, has been implicated in tumor cell proliferation and migration. There is a direct correlation between treatment-resistant breast carcinomas and the increased synthesis of HA via the over-expression of the HA anabolic enzyme, HA synthase (HAS). It is becoming generally accepted that the failure of cancer treatments can be due to a small subpopulation of slow growing, invasive and drug resistant cells known as cancer stem cells (CSCs) which are often characterized by the presence of CD44. As CD44 is the sole HA metabolic receptor within the tumor microenvironment, this study aimed to characterize the role of HA metabolism in the breast CSC subpopulation and its potential contribution to the persistence of treatment-resistant cancer. Methodology:HA metabolic enzymes [HAS 1-3 and hyaluronidase 1-2 (Hyal)] in breast cancer cell subpopulations were characterised in five cancer cell lines after FACS separation into putative CSC (CD44+/CD24-) and non-stem cell (CD44-/CD24+) subpopulations using quantitative real time PCR. Cell subpopulations were further characterised for cell-growth kinetics and resistance to chemotherapy using an Excelligence proliferation assay. The most highly expressed HAS isoform, HAS2, was knocked down or inserted in different cell lines using stable transfection and the resultant changes in proliferation, invasive and migratory potential and the direct effect on the CSC subpopulation was investigated both in vitro and in vivo. Results:HAS2 and Hyal2 were determined to be preferentially expressed in invasive breast cancer cell lines, where the chemotherapy-resistant CSC subpopulations (CD44+/CD24-) expressed the highest level of these HAS and Hyal isoforms. Transfection-induced attenuation of HAS2 mediated a reduction in the CSC subpopulation while over-expression of HAS2 increased the size of the CD44+/CD24- subpopulation. Both in vitro and in vivo HAS 2 over-expression increased HA production with a concomitant increase in tumorigenesis and metastasis while the counter-effect was observed in HAS2 knock-down breast cancer cells. Conclusions:These preliminary data suggest that HA metabolism is a tightly regulated process and a key component within breast cancer subpopulations and the CSC niche. The direct relationship between increased HAS expression, HA production and enlargement of the CD44+/CD24- subpopulation highlights HA metabolic enzymes as potential therapeutic targets in treatment-resistant breast cancer. Citation Format: Vera Evtimov, Tracey Brown. The characterisation of hyaluronan-related enzymes in breast cancer cell subpopulations. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3023. doi:10.1158/1538-7445.AM2014-3023

Divergent Temporal Expression of Hyaluronan Metabolizing Enzymes and Receptors with Craniotomy vs. Controlled-Cortical Impact Injury in Rat Brain: A Pilot Study.

Traumatic brain injury (TBI) triggers many secondary changes in tissue biology, which ultimately determine the extent of injury and clinical outcome. Hyaluronan [hyaluronic acid (HA)] is a protective cementing gel present in the intercellular spaces whose degradation has been reported as a causative factor in tissue damage. Yet little is known about the expression and activities of genes involved in HA catabolism after TBI. Young adult male Sprague-Dawley rats were assigned to three groups: naïve control, craniotomy, and controlled-cortical impact-induced TBI (CCI-TBI). Four animals per group were sacrificed at 4 h, 1, 3, and 7 days post-CCI. The mRNA expression of hyaluronan synthases (HAS1-3), hyaluronidases (enzymes for HA degradation, HYAL 1–4, and PH20), and CD44 and RHAMM (membrane receptors for HA signaling and removal) were determined using real-time PCR. Compared to the naïve controls, expression of HAS1 and HAS2 mRNA, but not HAS3 mRNA increased significantly following craniotomy alone and following CCI with differential kinetics. Expression of HAS2 mRNA increased significantly in the ipsilateral brain at 1 and 3 days post-CCI. HYAL1 mRNA expression also increased significantly in the craniotomy group and in the contralateral CCI at 1 and 3 days post-CCI. CD44 mRNA expression increased significantly in the ipsilateral CCI at 4 h, 1, 3, and 7 days post-CCI (up to 25-fold increase). These data suggest a dynamic regulation and role for HA metabolism in secondary responses to TBI.

Biological responses of three-dimensional cultured fibroblasts by sustained compressive loading include apoptosis and survival activity.

Pressure ulcers are characterized by chronicity, which results in delayed wound healing due to pressure. Early intervention for preventing delayed healing due to pressure requires a prediction method. However, no study has reported the prediction of delayed healing due to pressure. Therefore, this study focused on biological response-based molecular markers for the establishment of an assessment technology to predict delayed healing due to pressure. We tested the hypothesis that sustained compressive loading applied to three dimensional cultured fibroblasts leads to upregulation of heat shock proteins (HSPs), CD44, hyaluronan synthase 2 (HAS2), and cyclooxygenase 2 (COX2) along with apoptosis via disruption of adhesion. First, sustained compressive loading was applied to fibroblast-seeded collagen sponges. Following this, collagen sponge samples and culture supernatants were collected for apoptosis and proliferation assays, gene expression analysis, immunocytochemistry, and quantification of secreted substances induced by upregulation of mRNA and protein level. Compared to the control, the compressed samples demonstrated that apoptosis was induced in a time- and load- dependent manner; vinculin and stress fiber were scarce; HSP90α, CD44, HAS2, and COX2 expression was upregulated; and the concentrations of HSP90α, hyaluronan (HA), and prostaglandin E2 (PGE2) were increased. In addition, the gene expression of antiapoptotic Bcl2 was significantly increased in the compressed samples compared to the control. These results suggest that compressive loading induces not only apoptosis but also survival activity. These observations support that HSP90α, HA, and, PGE2 could be potential molecular markers for prediction of delayed wound healing due to pressure.

Hyaluronan Metabolism in Human Keratinocytes and Atopic Dermatitis Skin Is Driven by a Balance of Hyaluronan Synthases 1 and 3

Hyaluronan (HA) is a glycosaminoglycan synthesized directly into the extracellular matrix by three hyaluronan synthases (HAS1, HAS2, and HAS3). HA is abundantly synthesized by keratinocytes but its epidermal functions remain unclear. We used culture models to grow human keratinocytes as autocrine monolayers or as reconstructed human epidermis (RHE) to assess HA synthesis and HAS expression levels during the course of keratinocyte differentiation. In both the models, epidermal differentiation downregulates HAS3 mRNA expression while increasing HAS1 without significant changes in hyaluronidase expression. HA production correlates with HAS1 mRNA expression level during normal differentiation. To investigate the regulation of HAS gene expression during inflammatory conditions linked to perturbed differentiation, lesional and non-lesional skin biopsies of atopic dermatitis (AD) patients were analyzed. HAS3 mRNA expression level increases in AD lesions compared with healthy and non-lesional skin. Simultaneously, HAS1 expression decreases. Heparin-binding EGF-like growth factor (HB-EGF) is upregulated in AD epidermis. An AD-like HAS expression pattern is observed in RHE incubated with HB-EGF. These results indicate that HAS1 is the main enzyme responsible for HA production by normal keratinocytes and thus, must be considered as an actor of normal keratinocyte differentiation. In contrast, HAS3 can be induced by HB-EGF and seems mainly involved in AD epidermis.

Extracellular UDP-Glucose Activates P2Y14 Receptor and Induces Signal Transducer and Activator of Transcription 3 (STAT3) Tyr705 Phosphorylation and Binding to Hyaluronan Synthase 2 (HAS2) Promoter, Stimulating Hyaluronan Synthesis of Keratinocytes

Hyaluronan, a major matrix molecule in epidermis, is often increased by stimuli that enhance keratinocyte proliferation and migration. We found that small amounts of UDP-sugars were released from keratinocytes and that UDP-glucose (UDP-Glc) added into keratinocyte cultures induced a specific, rapid induction of hyaluronan synthase 2 (HAS2), and an increase of hyaluronan synthesis. The up-regulation of HAS2 was associated with JAK2 and ERK1/2 activation, and specific Tyr(705) phosphorylation of transcription factor STAT3. Inhibition of JAK2, STAT3, or Gi-coupled receptors blocked the induction of HAS2 expression by UDP-Glc, the latter inhibitor suggesting that the signaling was triggered by the UDP-sugar receptor P2Y14. Chromatin immunoprecipitations demonstrated increased promoter binding of Tyr(P)(705)-STAT3 at the time of HAS2 induction. Interestingly, at the same time Ser(P)(727)-STAT3 binding to its response element regions in the HAS2 promoter was unchanged or decreased. UDP-Glc also stimulated keratinocyte migration, proliferation, and IL-8 expression, supporting a notion that UDP-Glc signals for epidermal inflammation, enhanced hyaluronan synthesis as an integral part of it.

Metabolic engineering of Pichia pastoris for production of hyaluronic acid with high molecular weight

The high molecular weight (>1MDa) of hyaluronic acid (HA) is important for its biological functions. The reported limiting factors for the production of HA with high molecular weight (MW) by microbial fermentation are the insufficient HA precursor pool and cell growth inhibition. To overcome these issues, the Xenopus laevis xhasA2 and xhasB genes encoding hyaluronan synthase 2 (xhasA2) and UDP-glucose dehydrogenase (xhasB), were expressed in Pichia pastoris widely used for production of heterologous proteins. In this study, expression vectors containing various combination cassettes of HA pathway genes including xhasA2 and xhasB from X. laevis as well as UDP-glucose pyrophosphorylase (hasC), UDP-N-acetylglucosamine pyrophosphorylase (hasD) and phosphoglucose isomerase (hasE) from P. pastoris were constructed and tested. First, HA pathway genes were overexpressed using pAO815 and pGAPZB vectors, resulting in the production of 1.2MDa HA polymers. Second, in order to decrease hyaluronan synthase expression a strong AOX1 promoter in the xhasA2 gene was replaced by a weak AOX2 promoter which increased the mean MW of HA to 2.1MDa. Finally, the MW of HA polymer was further increased to 2.5MDa by low-temperature cultivation (26°C) which reduced cell growth inhibition. The yield of HA production by the P. pastoris recombinant strains in 1L of fermentation culture was 0.8–1.7g/L.

Cyclic phosphatidic acid and lysophosphatidic acid induce hyaluronic acid synthesis via CREB transcription factor regulation in human skin fibroblasts

Cyclic phosphatidic acid (cPA) is a naturally occurring phospholipid mediator and an analog of the growth factor-like phospholipid lysophosphatidic acid (LPA). cPA has a unique cyclic phosphate ring at the sn-2 and sn-3 positions of its glycerol backbone. We showed before that a metabolically stabilized cPA derivative, 2-carba-cPA, relieved osteoarthritis pathogenesis in vivo and induced hyaluronic acid synthesis in human osteoarthritis synoviocytes in vitro. This study focused on hyaluronic acid synthesis in human fibroblasts, which retain moisture and maintain health in the dermis. We investigated the effects of cPA and LPA on hyaluronic acid synthesis in human fibroblasts (NB1RGB cells). Using particle exclusion and enzyme-linked immunosorbent assays, we found that both cPA and LPA dose-dependently induced hyaluronic acid synthesis. We revealed that the expression of hyaluronan synthase 2 messenger RNA and protein is up-regulated by cPA and LPA treatment time dependently. We then characterized the signaling pathways up-regulating hyaluronic acid synthesis mediated by cPA and LPA in NB1RGB cells. Pharmacological inhibition and reporter gene assays revealed that the activation of the LPA receptor LPAR1, Gi/o protein, phosphatidylinositol-3 kinase (PI3K), extracellular-signal-regulated kinase (ERK), and cyclic adenosine monophosphate response element-binding protein (CREB) but not nuclear factor κB induced hyaluronic acid synthesis by the treatment with cPA and LPA in NB1RGB cells. These results demonstrate for the first time that cPA and LPA induce hyaluronic acid synthesis in human skin fibroblasts mainly through the activation of LPAR1-Gi/o followed by the PI3K, ERK, and CREB signaling pathway.