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Under physiological conditions in vivo astrocytes internalize and degrade neuronal mitochondria in a process called transmitophagy. Mitophagy is widely reported to be impaired in neurodegeneration but it is unknown whether and how transmitophagy is altered in Alzheimer’s disease (AD). Here we report that the internalization and degradation of neuronal mitochondria are significantly increased in astrocytes isolated from aged AD mouse brains. We also demonstrate for the first time a similar phenomenon between human neurons and AD astrocytes, and in murine hippocampi in vivo. The results suggest the involvement of S100a4 in impaired mitochondrial transfer between neurons and aged AD astrocytes. Significant increases in the mitophagy regulator Ambra1 were observed in the aged AD astrocytes. These findings demonstrate altered neuron-supporting functions of aged AD astrocytes and provide a starting point for studying the molecular mechanisms of transmitophagy in AD.

Filopodia are multifunctional finger-like plasma membrane protrusions with bundles of actin filaments that exist in virtually all cell types. It has been known for some time that hyaluronan synthesis activity induces filopodial growth. However, because of technical challenges in the studies of these slender and fragile structures, no quantitative analyses have been performed so far to indicate their association with hyaluronan synthesis. In this work we comprehensively address the direct quantification of filopodial traits, covering for the first time length and density measurements in a series of human cancer cell lines with variable levels of hyaluronan synthesis. The synthesis and plasma membrane binding of hyaluronan were manipulated with hyaluronan synthase 3 (HAS3) and hyaluronan receptor CD44 overexpression, and treatments with mannose, 4-methylumbelliferone (4-MU), and glucosamine. The results of this work show that the growth of filopodia was associated with the levels of hyaluronan synthesis but was not dependent on CD44 expression. The results confirm the hypothesis that abundance and length of filopodia in cancer cells is associated with the activity of hyaluronan synthesis.

The mesothelium is a membrane that forms the lining of several body cavities. It is composed of simple squamous mesothelial cells that secrete a glycosaminoglycan-rich lubricating fluid between inner organs. One of the most abundant glycosaminoglycans of those fluids is hyaluronan, which is synthesized on a plasma membrane and especially on apical filopodia of cultured cells. Our recent study showed that similar hyaluronan-rich protrusions are found in mesothelial lining in vivo, which suggests that hyaluronan synthesis in plasma membrane protrusions is a general process. However, the mesothelial lining was negative for the hyaluronan receptor CD44 while in many previous studies cultured mesothelial cells have been shown to express CD44. To further explore these findings we induced epithelial to mesenchymal transition in primary rat mesothelial cells by EGF-treatment and scratch wounding. Surprisingly, the results showed that at a normal epithelial, confluent stage the mesothelial cells are negative for CD44, but EMT induced by EGF or wounding activates CD44 expression and the whole hyaluronan synthesis machinery. In addition to typical EMT-like morphological changes, the growth of apical filopodia and budding of extracellular vesicles (EVs) were induced. In summary, the results of this study show that the activation of hyaluronan synthesis machinery, especially the expression of CD44 is strongly associated with EMT induced by EGF and wounding in mesothelial cells. Moreover, EMT enhances the secretion of EVs that carry CD44 and hyaluronan, which may be important regulators in EV interactions with their targets and ECM remodeling. The results of the present study also suggest that CD44 is a potential marker for EVs, especially those secreted from cells during tissue repair and pathological processes.

Background DHCR24, involved in the de novo synthesis of cholesterol and protection of neuronal cells against different stress conditions, has been shown to be selectively downregulated in neurons of the affected brain areas in Alzheimer’s disease. Methods Here, we investigated whether the overexpression of DHCR24 protects neurons against inflammation-induced neuronal death using co-cultures of mouse embryonic primary cortical neurons and BV2 microglial cells upon acute neuroinflammation. Moreover, the effects of DHCR24 overexpression on dendritic spine density and morphology in cultured mature mouse hippocampal neurons and on the outcome measures of ischemia-induced brain damage in vivo in mice were assessed. Results Overexpression of DHCR24 reduced the loss of neurons under inflammation elicited by LPS and IFN-γ treatment in co-cultures of mouse neurons and BV2 microglial cells but did not affect the production of neuroinflammatory mediators, total cellular cholesterol levels, or the activity of proteins linked with neuroprotective signaling. Conversely, the levels of post-synaptic cell adhesion protein neuroligin-1 were significantly increased upon the overexpression of DHCR24 in basal growth conditions. Augmentation of DHCR24 also increased the total number of dendritic spines and the proportion of mushroom spines in mature mouse hippocampal neurons. In vivo, overexpression of DHCR24 in striatum reduced the lesion size measured by MRI in a mouse model of transient focal ischemia. Conclusions These results suggest that the augmentation of DHCR24 levels provides neuroprotection in acute stress conditions, which lead to neuronal loss in vitro and in vivo., published version, peerReviewed

Intercellular communication is fundamental to the survival and maintenance of all multicellular systems, whereas dysregulation of communication pathways can drive cancer progression. Extracellular vesicles (EVs) are mediators of cell-to-cell communication that regulate a variety of cellular processes involved in tumor progression. Overexpression of a specific plasma membrane enzyme, hyaluronan synthase 3 (HAS3), is one of the factors that can induce EV shedding. HAS3, and particularly its product hyaluronan (HA), are carried by EVs and are known to be associated with the tumorigenic properties of cancer cells. To elucidate the specific effects of cancerous, HAS3-induced EVs on target cells, normal human keratinocytes and melanoma cells were treated with EVs derived from GFP-HAS3 expressing metastatic melanoma cells. We found that the HA receptor CD44 participated in the regulation of EV binding to target cells. Furthermore, GFP-HAS3-positive EVs induced HA secretion, proliferation and invasion of target cells. Our results suggest that HAS3-EVs contains increased quantities of IHH, which activates the target cell hedgehog signaling cascade and leads to the activation of c-Myc and regulation of claspin expression. This signaling of IHH in HAS3-EVs resulted in increased cell proliferation. Claspin immunostaining correlated with HA content in human cutaneous melanocytic lesions, supporting our in vitro findings and suggesting a reciprocal regulation between claspin expression and HA synthesis. This study shows for the first time that EVs originating from HAS3 overexpressing cells carry mitogenic signals that induce proliferation and epithelial-to-mesenchymal transition in target cells. The study also identifies a novel feedback regulation between the hedgehog signaling pathway and HA metabolism in melanoma, mediated by EVs carrying HA and IHH. Electronic supplementary material The online version of this article (10.1007/s00018-019-03399-5) contains supplementary material, which is available to authorized users.

Extracellular vesicles (EVs) and their interactions with recipient cells constitute a rapidly growing research area. However, due to the limitations in current methodologies, the mechanisms of these interactions are still unclear. Microscopic studies of EVs are challenging, because their typical diameter is near the resolution limit of light microscopy, and electron microscopy has restricted possibilities for protein labelling. The objective of this study was to combine these two techniques to demonstrate in detail the interactions of EVs by recipient cells. Hyaluronan synthase 3 (HAS3) is an integral transmembrane protein that is enriched in EVs. In this work, GFP-HAS3 was utilized to study the interactions of EVs with the recipient cells. Surprisingly, confocal analysis correlation with scanning electron microscopy (SEM) revealed that most of the EVs were indeed lying on the recipient cell's plasma membrane, while the level of EV-derived intracellular signal was low. Immunoelectron microscopy supported this finding. Furthermore, hyaluronan oligosaccharides decreased the numbers of bound EVs, suggesting that CD44 participates in the regulation of their binding. This study indicates that correlative light and electron microscopy is a reliable method to analyze EV interactions with recipient cells. Detailed 3D confocal imaging of EV carrying a GFP-label on their plasma membrane combined with high-resolution electron microscopy provides significantly more information than either of the techniques alone. In the future studies it is crucial to utilize these techniques and their combinations to solve in detail the ambiguous fate of EV in target cells. Furthermore, live cell imaging at high resolution will be required to obtain definite answers on the detailed mechanisms of binding, fusion and endocytosis of EVs.

Obesity and oversupply of glucose, e.g., due to nutritional factors may shape the tumor microenvironment favorable for tumor progression. O-GlcNAcylation, a reversible modification of intracellular proteins, influences on several cellular functions and is connected to many diseases including cancer. Glycosaminoglycan hyaluronan (HA) enhances tumor progression and in breast cancer HA accumulation associates strongly with poor outcome. In vitro studies have suggested that O-GlcNAcylation may enhance HA synthesis. The aim of this study was to investigate the correlations between O-GlcNAcylation, HA-related parameters, and disease outcome in a clinical breast cancer material consisting of 278 breast cancer cases. In microscopic analyses, O-GlcNAc staining of the breast carcinoma cells was evaluated in several randomly picked high-power fields of each section. The extent of cytoplasmic O-GlcNAc staining was graded as either low or high according to the intensity of the staining and the percentage of stained cells. The extent of nuclear O-GlcNAc staining was categorized as either low or high according to the percentage of stained nuclei. A high extent of both cytoplasmic and nuclear O-GlcNAcylation correlated with an increased relapse rate, development of distant metastases, and poor outcome. A high extent of cytoplasmic O-GlcNAcylation correlated also with the accumulation of all hyaluronan synthase (HAS1-3) proteins and with a large amount of HA in the tumor stroma. In addition, a high extent of nuclear O-GlcNAcylation associated with obesity. The results suggest a mechanistic association between increased O-GlcNAcylation and HA synthesis, leading to a HA-rich microenvironment favorable for breast cancer progression.

Article, Previous studies have shown that overexpression of enzymatically active GFP-HAS induces the growth of long, slender protrusions that share many features of both filopodia and microvilli. These protrusions are dependent on continuing hyaluronan synthesis, and disrupt upon digestion of hyaluronan by hyaluronidase. However, complete understanding of their nature is still missing. This work shows that the protrusions on rat peritoneal surface are ultrastructurally indistinguishable from those induced by GFP-HAS3 in MCF-7 cells. Analysis of the actin-associated proteins villin, ezrin, espin, fascin, and Myo10 indicated that the HAS3-induced protrusions share most cytoskeletal features with filopodia, but they do not require adherence to the substratum like traditional filopodia. GFP-HAS3 overexpression was found to markedly enhance filamentous actin in the protrusions and their cortical basis. Analysis of the protrusion dynamics after enzymatic digestion of hyaluronan revealed that while GFP-HAS3 escape from the protrusions and the protrusion collapse takes place immediately, the complete retraction of the protrusions occurs more slowly. This finding also suggests that hyaluronan chain maintains HAS3 in the plasma membrane. The results of this work suggest that protrusions similar to those of HAS3 overexpressing cells in vitro exist also in cells with active hyaluronan synthesis in vivo. These protrusions are similar to common filopodia but are independent of substratum attachment due to the extracellular scaffolding by the hyaluronan coat that accounts for the growth and maintenance of these structures, previously associated to invasion, adhesion and multidrug resistance., final draft, http://purl.org/eprint/status/PeerReviewed

PURPOSE. Retinal pigment epithelium (RPE) limits the xenobiotic entry from the systemic blood stream to the eye. RPE surface transporters can be important in ocular drug distribution, but it has been unclear whether they are expressed on the apical, basal, or both cellular surfaces. In this paper, we provide quantitative comparison of apical and basolateral RPE surface proteomes. METHODS. We separated the apical and basolateral membranes of differentiated human fetal RPE (hfRPE) cells by combining apical membrane peeling and sucrose density gradient centrifugation. The membrane fractions were analyzed with quantitative targeted absolute proteomics (QTAP) and sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH-MS) to reveal the membrane protein localization on the RPE cell surfaces. We quantitated 15 transporters in unfractionated RPE cells and scaled their expression to tissue level. RESULTS. Several proteins involved in visual cycle, cell adhesion, and ion and nutrient transport were expressed on the hfRPE plasma membranes. Most drug transporters showed similar abundance on both RPE surfaces, whereas large neutral amino acids transporter 1 (LAT1), p-glycoprotein (P-gp), and monocarboxylate transporter 1 (MCT1) showed modest apical enrichment. Many solute carriers (SLC) that are potential prodrug targets were present on both cellular surfaces, whereas putative sodium-coupled neutral amino acid transporter 7 (SNAT7) and riboflavin transporter (RFT3) were enriched on the basolateral and sodium- and chloride-dependent neutral and basic amino acid transporter (ATB(0+)) on the apical membrane. CONCLUSIONS. Comprehensive quantitative information of the RPE surface proteomes was reported for the first time. The scientific community can use the data to further increase understanding of the RPE functions. In addition, we provide insights for transporter protein localization in the human RPE and the significance for ocular pharmacokinetics.

Vesicular trafficking of hyaluronan synthases (HAS1-3) from endoplasmic reticulum (ER) through Golgi to plasma membrane (PM), and either back to endosomes and lysosomes, or out into extracellular vesicles, is important for their activities. We studied how post-translational modifications affect the trafficking of HAS2 by mutagenesis of the sites of ubiquitination (K190R), phosphorylation (T110A) and O-GlcNAcylation (S221A), using Dendra2- and EGFP-HAS2 transfected into COS1 cells. Confocal microscopy showed HAS2 wild type (wt) and its K190R and S221A mutants in ER, Golgi and extracellular vesicles, while the T110A mutant remained mostly in the ER. HA synthesis was reduced by S221A, while completely blocked by K190R and T110A. Cell-surface biotinylation indicated that T110A was absent from PM, while S221A was close to the level of wt, and K190R was increased in PM. TIRF microscopy analysis gave similar results. Rab10 silencing increased HA secretion by HAS2, likely by inhibiting endocytosis of the enzyme from PM, as reported before for HAS3. Green-to-red photo-conversion of Dendra2-HAS2 constructs suggested slower decay of K190R and S221A than HAS2 wt, while T110A was barely degraded at all. S221D and S221E, the phosphomimetic mutants of this site, decayed faster and blocked hyaluronan synthesis, suggesting alternative O-GlcNAc/-PO4 substitution to regulate the stability of the enzyme. Probing the role of dynamic O-GlcNAcylation at S221 by adding glucosamine increased the half-life of only HAS2 wt. The Dendra2·HAS2 disappearance from Golgi was slower for K190R. Of the two inactive constructs, K190R co-transfected with HAS2 wt suppressed, whereas T110A had no effect on HA synthesis. Interestingly, the HAS2-stimulated shedding of extracellular vesicles was dependent on HAS residence in PM but independent of HA synthesis. The results indicate that post-translational modifications control the trafficking of HAS2, and that trafficking is an integral part of the post-translational regulation of HAS2 activity.

Background Diffusely infiltrating astrocytomas originate from astrocytic glial cells or their precursor cells and are the most common type of brain tumors in adults. In this retrospective study, we investigated the content of hyaluronan, its cell surface receptor, CD44 and the expression of hyaluronan metabolizing enzymes, in these aggressive tumors. Hyaluronan is the main component of extracellular matrix in the brain. In many tumors, aberrant hyaluronan metabolism implicates aggressive disease progression and metastatic potential. Methods Our material consisted of 163 diffusely infiltrating astrocytomas (WHO grades II-IV). Tumor samples were processed into tissue microarray (TMA) blocks. The TMA sections were stained for hyaluronan, CD44, hyaluronan synthases 1–3 (HAS1–3) and hyaluronidase 2 (HYAL2). The immunostaining results were compared with χ2 –test or with Kruskal-Wallis test for correlation with clinicopathological parameters and survival analyses were done with Kaplan-Meier log rank test and Cox regression. Results Hyaluronan and CD44 were strongly expressed in astrocytic gliomas but their expression did not correlate with WHO grade or any other clinicopathological parameters whereas high HAS2 staining intensity was observed in IDH1 negative tumors (p = 0.003). In addition, in non-parametric tests increased HAS2 staining intensity correlated with increased cell proliferation (p = 0.013) and in log rank test with decreased overall survival of patients (p = 0.001). In the Cox regression analysis HAS2 expression turned out to be a significant independent prognostic factor (p = 0.008). Conclusions This study indicates that elevated expression of HAS2 is associated with glioma progression and suggests that HAS2 has a prognostic significance in diffusely infiltrating astrocytomas. Electronic supplementary material The online version of this article (10.1186/s12885-018-4569-1) contains supplementary material, which is available to authorized users.

Malignant skin melanoma is one of the most deadly human cancers. Extracellular matrix (ECM) influences the growth of malignant tumors by modulating tumor cells adhesion and migration. Hyaluronan is an essential component of the ECM, and its amount is altered in many tumors, suggesting an important role for hyaluronan in tumorigenesis. Nonetheless its role in melanomagenesis is not understood. In this study we produced a MV3 melanoma cell line with inducible expression of the hyaluronan synthase 3 (HAS3) and studied its effect on the behavior of the melanoma cells. HAS3 overexpression expanded the cell surface hyaluronan coat and decreased melanoma cell adhesion, migration and proliferation by cell cycle arrest at G1/G0. Melanoma cell migration was restored by removal of cell surface hyaluronan by Streptomyces hyaluronidase and by receptor blocking with hyaluronan oligosaccharides, while the effect on cell proliferation was receptor independent. Overexpression of HAS3 decreased ERK1/2 phosphorylation suggesting that inhibition of MAP-kinase signaling was responsible for these suppressive effects on the malignant phenotype of MV3 melanoma cells.

The proinflammatory cytokine interleukin-1β (IL-1β) attracts leukocytes to sites of inflammation. One of the recruitment mechanisms involves the formation of extended, hyaluronan-rich pericellular coats on local fibroblasts, endothelial cells, and epithelial cells. In the present work, we studied how IL-1β turns on the monocyte adhesion of the hyaluronan coat on human keratinocytes. IL-1β did not influence hyaluronan synthesis or increase the amount of pericellular hyaluronan in these cells. Instead, we found that the increase in the hyaluronan-dependent monocyte binding was associated with the CD44 of the keratinocytes. Although IL-1β caused a small increase in the total amount of CD44, a more marked impact was the decrease of CD44 phosphorylation at serine 325. At the same time, IL-1β increased the association of CD44 with ezrin and complex formation of CD44 with itself. Treatment of keratinocyte cultures with KN93, an inhibitor of calmodulin kinase 2, known to phosphorylate Ser-325 in CD44, caused similar effects as IL-1β (i.e. homomerization of CD44 and its association with ezrin) and resulted in increased monocyte binding to keratinocytes in a hyaluronan-dependent way. Overexpression of wild type CD44 standard form, but not a corresponding CD44 mutant mimicking the Ser-325-phosphorylated form, was able to induce monocyte binding to keratinocytes. In conclusion, treatment of human keratinocytes with IL-1β changes the structure of their hyaluronan coat by influencing the amount, post-translational modification, and cytoskeletal association of CD44, thus enhancing monocyte retention on keratinocytes.

Aims High amounts of tumour-associated macrophages (TAMs) and hyaluronan (HA) correlate with tumour aggressiveness in breast cancer, but the relationship between these parameters is unclear. The aim of this study was to assay the numbers of TAMs in 278 human breast cancer cases, and their correlations with HA-related factors, clinical variables, and outcome. Methods and results The immunoreactivities for CD163 and CD68 were considered as indicators for M2-like and all TAMs, respectively. The numbers of TAMs were counted in at least four hot spots, and averaged to represent the numbers of TAMs in each section. In the statistical analyses, the numbers were graded as either low or high according to the median. High numbers of TAMs correlated with a high tumour HA content, HA synthases, CD44 positivity, and poor outcome. The number of CD163-positive cells represented a strong independent prognostic factor. There was also a significant correlation between obesity and a high number of CD163-positive cells. Conclusions Concurrent increases in TAMs and HA in breast cancer indicate that the accumulation of HA facilitates macrophage infiltration and inflammatory responses during human breast cancer progression.

Taurine is a small organic osmolyte which participates in cell volume regulation. Chondrocytes have been shown to accumulate and release taurine; in bone, taurine participates in bone metabolism. However, its role in skeletal cells is poorly understood, especially in chondrocytes. This study investigated the regulation of taurine transporter in chondrocytic cells. We examined the transcriptional regulation of the taurine transporter under anisotonia by reporter gene and real-time RT-PCR assays. The effect of providing supplementary taurine on cell viability was evaluated with the lactate dehydrogenase release assay. The localization of the taurine transporter in human chondrosarcoma cells was studied by overexpressing a taurine transporter-enhanced green fluorescent protein. We observed that the transcription of the taurine transporter gene was up-regulated in hypertonic conditions. Hyperosmolarity-related cell death could be partly abolished by taurine supplementation in the medium. As expected, the fluorescently labeled taurine transporter localized at the plasma membrane. In polarized epithelial MDCK cells, the strongest fluorescence signal was located in the lateral cell membrane area. We also observed that the taurine transporter gene was expressed in several human tissues and malignant cell lines. This is the first study to present information on the transcriptional regulation of taurine transporter gene and the localization of the taurine transporter protein in chondrocytic cells.

Extracellular vesicles (EV) are small plasma membrane-derived particles released into the extracellular space by virtually all cell types. Recently, EV have received increased interest because of their capability to carry nucleic acids, proteins, lipids and signaling molecules and to transfer their cargo into the target cells. Less attention has been paid to their role in modifying the composition of the extracellular matrix (ECM), either directly or indirectly via regulating the ability of target cells to synthesize or degrade matrix molecules. Based on recent results, EV can be considered one of the structural and functional components of the ECM that participate in matrix organization, regulation of cells within it, and in determining the physical properties of soft connective tissues, bone, cartilage and dentin. This review addresses the relevance of EV as specific modulators of the ECM, such as during the assembly and disassembly of the molecular network, signaling through the ECM and formation of niches suitable for tissue regeneration, inflammation and tumor progression. Finally, we assess the potential of these aspects of EV biology to translational medicine.

Extracellular vesicles (EVs) secreted by stem cells are potential factors mediating tissue regeneration. They travel from bone marrow stem cells into damaged tissues, suggesting that they can repair tissue injuries without directly replacing parenchymal cells. We have discovered that hyaluronan (HA) synthesis is associated with the shedding of HA-coated EVs. The aim of this study was to test whether bone marrow-derived hMSCs secrete HA-coated EVs. The EVs secreted by MSCs were isolated by differential centrifugation and characterized by nanoparticle tracking analysis. Their morphology and budding mechanisms were inspected by confocal microscopy and correlative light and electron microscopy. Hyaluronan synthesis of hMSCs was induced by lipopolysaccharide and inhibited by RNA interference and 4-methylumbelliferone. It was found that the MSCs have extremely long apical and lateral HA-coated filopodia, typical for cells with an active HA secretion. Additionally, they secreted HA-coated EVs carrying mRNAs for CD44 and all HAS isoforms. The results show that stem cells have a strong intrinsic potential for HA synthesis and EV secretion, and the amount of HA carried on EVs reflects the HA content of the original cells. These results show that the secretion of HA-coated EVs by hMSCs is a general process, that may contribute to many of the mechanisms of HA-mediated tissue regeneration. Additionally, an HA coat on EVs may regulate their interactions with target cells and participate in extracellular matrix remodeling.

Chloropicrin is a vaporizing, irritating compound that causes complications in the respiratory system when inhaled. In this study, we examined the effects of exposure to chloropicrin for 24 h on ultrastructure and global gene expression in primary human bronchial epithelial cells. The treatment increased the number of round and shrunken cells, which detached from culture plates more readily than the untreated control cells. Transmission electron microscopy revealed some swollen mitochondria and the appearance of autophagy/lysosome type of vacuoles in the treated cells. However, the main alteration in the ultrastructure of the treated cells was the presence of aggregated and slightly deformed cytoskeleton structures. Furthermore, confocal microscopy and immunoblotting indicated that cytoskeletal β-tubulin protein is a probable target of chloropicrin exposure. Ingenuity Pathway Analysis (IPA) of differentially expressed microarray data (fold change > ± 2 compared to controls considered) revealed that the top molecular functions were cell growth and proliferation. The main enriched top canonical pathways identified by IPA were associated with EIF2-signalling, protein ubiquitination pathway, glycolysis and mitochondrial dysfunction. Furthermore, the main upstream regulators and their target genes were involved in cell growth and proliferation and cytoskeletal organization. The alterations found here can be the core components of toxicity involved in the lung complications after chloropicrin exposure., final draft, peerReviewed

Accumulation of hyaluronan (HA) in pericellular stroma and carcinoma cells is predictive of unfavorable patient prognosis in many epithelial cancers. However, it is not known whether the HA originates from carcinoma or stromal cells, or whether increased expression of hyaluronan synthase proteins (HAS1-3) contributes to HA accumulation. In this study, localization and expression of HAS1-3 were evaluated immunohistochemically in 278 cases of human breast cancer, and correlated with prognostic factors and patient outcome. Both carcinoma cells and stromal cells were HAS-positive. In carcinoma cells, HAS1 and HA stainings correlated with each other, and HAS1 associated with estrogen receptor negativity, HER2 positivity, high relapse rate, and short overall survival. In stromal cells, the staining levels of all HAS isoforms correlated with the stromal HA staining, stromal cell CD44, high relapse rate, and short overall survival of the patients. In addition, expression levels of stromal HAS1 and HAS2 were related to obesity, large tumor size, lymph node positivity, and estrogen receptor negativity. Thus, stromal HAS1 and HAS3 were independent prognostic factors in the multivariate analysis. The data suggest that increased levels of HAS enzymes contribute to the accumulation of HA in breast cancer, and that HA is synthesized in carcinoma cells and stromal cells. The study also indicates that HAS enzyme levels are related to tumor aggressiveness and poor patient outcome representing potential targets for therapy.

1. Currently available in vitro blood-brain barrier models all have recognized restrictions. In addition to leakiness, inconsistent data about P-glycoprotein mediated efflux limit the attractiveness of the primary bovine brain microvessel endothelial cells (BBMECs). Therefore, we re-evaluated the role of P-glycoprotein mediated efflux with two culture conditions in BBMECs for prediction of drug permeability of potential P-glycoprotein substrates. 2. BBMECs were monocultured on filters on petri dishes and on filter inserts, and expression and localization of P-glycoprotein were compared by using western blot and confocal microscopy, respectively. The functionality of P-glycoprotein was assessed by using cellular uptake, calcein-AM and bidirectional transport assays. 3. P-glycoprotein expression was higher in BBMECs cultured on filter inserts decreasing the permeability of digoxin and paclitaxel, but not the permeability of vinblastine. However, the monocultured BBMECs were not able to demonstrate efflux in the bidirectional transport assays. Under certain culture conditions, occludin may not be correctly located, perhaps explaining in part the leakiness of BBMECs. 4. In conclusion, BBMECs, despite possessing a functional P-glycoprotein, under certain culture conditions may not be a suitable in vitro model for the bidirectional transport assays and for predicting the permeability of drugs and xenobiotics that are potential P-glycoprotein substrates.

Many cell types secrete plasma membrane-bound microvesicles, suggested to play an important role in tissue morphogenesis, wound healing, and cancer spreading. However, the mechanisms of their formation have remained largely unknown. It was found that the tips of long microvilli induced in cells by overexpression of hyaluronan synthase 3 (HAS3) were detach into the culture medium as microvesicles. Moreover, several cell types with naturally active hyaluronan synthesis released high numbers of plasma membrane-derived vesicles, and inhibition of hyaluronan synthesis reduced their formation. The vesicles contained HAS, and were covered with a thick hyaluronan coat, a part of which was retained even after purification with high-speed centrifugation. HAS3 overexpressing MDCK cells cultured in a 3-D matrix as epithelial cysts released large amounts of HAS- and hyaluronan-positive vesicles from their basal surfaces into the extracellular matrix. As far as we know, hyaluronan synthesis is one of the first molecular mechanisms shown to stimulate the production of microvesicles. The microvesicles have a potential to deliver the hyaluronan synthase machinery and membrane and cytoplasmic materials to other cells, influencing tissue regeneration, inflammation and tumor progression.

Aneuploidy, deviation from the normal chromosome number, and other chromosomal aberrations are commonly observed in cancer. Integrin-mediated adhesion and dynamic turnover of adhesion sites are required for successful cytokinesis of normal adherent cells and impaired cell division can lead to the generation of cells with abnormal chromosome contents. We find that repeated cytokinesis failure, due to impaired integrin traffic alone, is sufficient to induce chromosome aberrations resulting in the generation of aneuploid cells with malignant properties. Here, we have compared isogenic aneuploid and euploid cell lines with unravel aneuploidy-induced changes in cellular signaling. Euploid, non-transformed, and aneuploid, transformed, cell lines were investigated using genome-wide gene expression profiling, analysis of deregulated biological pathways and array-comparative genomic hybridization. We find that aneuploidy drives malignancy via inducing marked changes in gene and micro RNA expression profiles and thus imposing specific growth and survival promoting alterations in cellular signaling. Importantly, we identify Twist2 as a key regulator of survival, invasion and anchorage-independent growth in the aneuploid cells. In addition, alterations in lipid biosynthetic pathways and miR-10b upregulation are likely contributors to the malignant phenotype.

Hyaluronan (HA) is a large glycosaminoglycan produced by hyaluronan synthases (HAS), enzymes normally active at plasma membrane. While HA is delivered into the extracellular space, intracellular HA is also seen, mostly in vesicular structures, but there are also reports on its presence in the cytosol and specific locations and functions there. We probed the possibility of HA localization and functions in cytosol by microinjecting fluorescent HA binding complex (fHABC), HA fragments and hyaluronidase (HYAL) into cytosol. Microinjection of fHABC did not reveal HA-specific intracellular binding sites. Likewise, specific cytosolic binding sites for HA were not detected, as microinjected fluorescent HA composed of 4-8 monosaccharide units (HA4-HA8) were evenly distributed throughout the cells, including the nucleus, but excluded from membrane-bound organelles. The largest HA tested (∼HA120 or ∼25 kDa) did not enter the nucleus, and HA10-HA28 were progressively excluded from parts of nuclei resembling nucleoli. In contrast, HA oligosaccharides endocytosed from medium remained in vesicular compartments. The activity of HA synthesis was estimated by measuring the HA coat on green fluorescent protein (GFP)-HAS3-transfected MCF-7 cells. Microinjection of HA4 reduced coat size at 4 h, but increased at 24 h after injection, while larger HA-oligosaccharides and HYAL had no influence. As a positive control, microinjection of glucose increased coat size. In summary, no evidence for the presence or function of HA in cytosol was obtained. Also, the synthesis of HA and the active site of HAS were not accessible to competition, binding and degradation by cytosolic effectors, while synthesis responded to increased substrate supply.

Previous observations of our research group showed that HAS2 and HAS3 overexpression in cultured cells induces the formation of long and numerous microvillus-like cell protrusions, which are present also in cultured cell types with naturally high hyaluronan secretion and the cell protrusions resemble those found in mesothelial cells. The aim of this study was to investigate whether these hyaluronan secreting, actin-dependent protrusions exist also in vivo. It was found that rat mesothelium in vivo is positive for hyaluronan and Has1-3. Also microvilli in rat mesothelium and live primary cultures of mesothelial cells were found to be hyaluronan positive, and the cells expressed all Has isoforms. Furthermore, ultrastructure of the cell protrusions in rat mesothelium was similar to that induced by overexpression of HAS2 and HAS3, and the number and orientation of actin filaments supporting the cell protrusions was identical. The results of this study show that HA-positive protrusions exist in vivo and support the idea that hyaluronan secretion from plasma membrane protrusions is a general process. This mechanism is potentially crucial for the normal function and maintenance of tissues and body fluids and may be utilized in many therapeutic applications.

In many cancers hyaluronan content is increased, either by tumor cells or the surrounding stromal cells and this increased hyaluronan content correlates with unfavorable clinical prognosis. In the present work, we studied the effects of melanoma cell (aggressive melanoma cell line C8161)-derived factors on fibroblast hyaluronan synthesis, intracellular signaling, MMP expression and invasion. Treatment of the fibroblast cultures with melanoma cell conditioned medium (CM) caused accumulation of hyaluronan in the culture medium and formation of thick pericellular hyaluronan coat and hyaluronan cables. The expression of Has2 was increased approximately 20-fold by the C8161 melanoma cell CM, while Has1 and Has3 were increased twofold. Knock-down of Has2 expression with siRNA showed that Has2 was responsible for the increased hyaluronan synthesis induced by the melanoma cell CM. To find out the signaling routes, which led to Has2 upregulation, the phosphorylation profiles of 46 kinases were screened with phosphokinase array kit. Melanoma cell CM treatment strongly induced a rapid phosphorylation of p38, JNK, AKT, CREB, HSP27, STAT3 and cJUN. Treatment of the fibroblasts with specific inhibitors of PI3K, AKT and p38 reduced the melanoma cell CM-induced hyaluronan secretion, while the inhibitor of PDGFR totally blocked it. In addition, siRNA for PDGFRα/β inhibited Has2 upregulation in melanoma cell CM-treated fibroblasts. In parallel with the increased hyaluronan synthesis the melanoma cell CM-treated fibroblasts showed spindle shape, numerous long cell protrusions, enhanced MMP expression and increased invasion into collagen-Cultrex matrix. siRNA blocking of Has2 or PDGFRα/β expression reversed the stimulatory effect of melanoma cell CM on fibroblast invasion. PDGF secreted by melanoma cells thus mediated fibroblasts activation, with HAS2 upregulation as a major factor in the fibroblast response. This effect on stromal matrix is suggested to favor tumor growth.

Hyaluronan content is a powerful prognostic factor in many cancer types, but the molecular basis of its synthesis in cancer still remains unclear. Hyaluronan synthesis requires the transport of hyaluronan synthases (HAS1-3) from Golgi to plasma membrane (PM), where the enzymes are activated. For the very first time, the present study demonstrated a rapid recycling of HAS3 between PM and endosomes, controlled by the cytosolic levels of the HAS substrates UDP-GlcUA and UDP-GlcNAc. Depletion of UDP-GlcNAc or UDP-GlcUA shifted the balance towards HAS3 endocytosis, and inhibition of hyaluronan synthesis. In contrast, UDP-GlcNAc surplus suppressed endocytosis and lysosomal decay of HAS3, favoring its retention in PM, stimulating hyaluronan synthesis, and HAS3 shedding in extracellular vesicles. The concentration of UDP-GlcNAc also controlled the level of O-GlcNAc modification of HAS3. Increasing O-GlcNAcylation reproduced the effects of UDP-GlcNAc surplus on HAS3 trafficking, while its suppression showed the opposite effects, indicating that O-GlcNAc signaling is associated to UDP-GlcNAc supply. Importantly, a similar correlation existed between the expression of GFAT1 (the rate limiting enzyme in UDP-GlcNAc synthesis) and hyaluronan content in early and deep human melanomas, suggesting the association of UDP-sugar metabolism in initiation of melanomagenesis. In general, changes in glucose metabolism, realized through UDP-sugar contents and O-GlcNAc signaling, are important in HAS3 trafficking, hyaluronan synthesis, and correlates with melanoma progression.

CD44 is a ubiquitous cell surface glycoprotein, involved in important cellular functions including cell adhesion, migration, and modulation of signals from cell surface receptors. While most of these CD44 functions are supposed to involve hyaluronan, relatively little is known about the contribution of CD44 to hyaluronan maintenance and organization on cell surface, and the role of CD44 in hyaluronan synthesis and catabolism. Blocking hyaluronan binding either by CD44 antibodies, CD44-siRNA or hyaluronan decasaccharides (but not hexasaccharides) removed most of the hyaluronan from the surfaces of both human (HaCaT) and mouse keratinocytes, resembling results on cells from CD44⁻/⁻ animals. In vitro, compromising CD44 function led to reduced and increased amounts, respectively, of intracellular and culture medium hyaluronan, and specific accumulation below the cells. In vivo, CD44-deficiency caused no marked differences in hyaluronan staining intensity or localization in the fetal skin or in adult ear skin, while tail epidermis showed a slight reduction in epidermal hyaluronan staining intensity. However, CD44-deficient tail skin challenged with retinoic acid or tape stripping revealed diffuse accumulation of hyaluronan in the superficial epidermal layers, normally negative for hyaluronan. Our data indicate that CD44 retains hyaluronan in the keratinocyte pericellular matrix, a fact that has not been shown unambiguously before, and that hyaluronan abundance in the absence of CD44 can result in hyaluronan trapping in abnormal locations possibly interfering there with normal differentiation and epidermal barrier function.

Hyaluronan, a ubiquitous high-molecular-mass glycinoglycan on cell surfaces and in extracellular matrices, has a number of specific signaling functions in cell–cell communication. Changes in its content, molecular mass and turnover rate are crucial for cell proliferation, migration and apoptosis, processes that control tissue remodeling during embryonic development, inflammation, injury and cancer. To maintain tissue homeostasis, the synthesis of hyaluronan must therefore be tightly controlled. In this review, we highlight some recent data on the transcriptional regulation of hyaluronan synthase (Has1–3) expression and on the post-transcriptional control of hyaluronan synthase activity, which, in close association with the supply of the UDP-sugar substrates of hyaluronan synthase, adjust the rate of hyaluronan synthesis.

Hyaluronan synthases (HAS1-3) are integral plasma membrane proteins that synthesize hyaluronan, a cell surface and extracellular matrix polysaccharide necessary for many biological processes. It has been shown that HAS is partly localized in cholesterol-rich lipid rafts of MCF-7 cells, and cholesterol depletion with methyl-beta-cyclodextrin (MbetaCD) suppresses hyaluronan secretion in smooth muscle cells. However, the mechanism by which cholesterol depletion inhibits hyaluronan production has remained unknown. We found that cholesterol depletion from MCF-7 cells by MbetaCD inhibits synthesis but does not decrease the molecular mass of hyaluronan, suggesting no major influence on HAS stability in the membrane. The inhibition of hyaluronan synthesis was not due to the availability of HAS substrates UDP-GlcUA and UDP-GlcNAc. Instead, MbetaCD specifically down-regulated the expression of HAS2 but not HAS1 or HAS3. Screening of signaling proteins after MbetaCD treatment revealed that phosphorylation of Akt and its downstream target p70S6 kinase, both members of phosphoinositide 3-kinase-Akt pathway, were inhibited. Inhibitors of this pathway suppressed hyaluronan synthesis and HAS2 expression in MCF-7 cells, suggesting that the reduced hyaluronan synthesis by MbetaCD is due to down-regulation of HAS2, mediated by the phosphoinositide 3-kinase-Akt-mTOR-p70S6K pathway.

Article, Hyaluronan synthase 1 (HAS1) is one of three isoenzymes responsible for cellular hyaluronan synthesis. Interest in HAS1 has been limited because its role in hyaluronan production seems to be insignificant compared to the two other isoenzymes, HAS2 and HAS3, which have higher enzymatic activity. Furthermore, in most cell types studied so far, the expression of its gene is low and the enzyme requires high concentrations of sugar precursors for hyaluronan synthesis, even when overexpressed in cell cultures. Both expression and activity of HAS1 are induced by pro-inflammatory factors like interleukins and cytokines, suggesting its involvement in inflammatory conditions. Has1 is upregulated in states associated with inflammation, like atherosclerosis, osteoarthritis, and infectious lung disease. In addition, both full length and splice variants of HAS1 are expressed in malignancies like bladder and prostate cancers, multiple myeloma, and malignant mesothelioma. Interestingly, immunostainings of tissue sections have demonstrated the role of HAS1 as a poor predictor in breast cancer, and is correlated with high relapse rate and short overall survival. Utilization of fluorescently tagged proteins has revealed the intracellular distribution pattern of HAS1, distinct from other isoenzymes. In all cell types studied so far, a high proportion of HAS1 is accumulated intracellularly, with a faint signal detected on the plasma membrane and its protrusions. Furthermore, the pericellular hyaluronan coat produced by HAS1 is usually thin without induction by inflammatory agents or glycemic stress and depends on CD44–HA interactions. These specific interactions regulate the organization of hyaluronan into a leukocyte recruiting matrix during inflammatory responses. Despite the apparently minor enzymatic activity of HAS1 under normal conditions, it may be an important factor under conditions associated with glycemic stress like metabolic syndrome, inflammation, and cancer., published version, http://purl.org/eprint/status/PeerReviewed

Article, Nucleoli are not only organelles that produce ribosomal subunits. They are also overarching sensors of different stress conditions and they control specific nucleolar stress pathways leading to stabilization of p53. During DNA replication, ATR and its activator TopBP1 initiate DNA damage response upon DNA damage and replication stress. We found that a basal level of TopBP1 protein associates with ribosomal DNA repeat. When upregulated, TopBP1 concentrates at the ribosomal chromatin and initiates segregation of nucleolar components—the hallmark of nucleolar stress response. TopBP1-induced nucleolar segregation is coupled to shut-down of ribosomal RNA transcription in an ATR-dependent manner. Nucleolar segregation induced by TopBP1 leads to a moderate elevation of p53 protein levels and to localization of activated p53 to nucleolar caps containing TopBP1, UBF and RNA polymerase I. Our findings demonstrate that TopBP1 and ATR are able to inhibit the synthesis of rRNA and to activate nucleolar stress pathway; yet the p53-mediated cell cycle arrest is thwarted in cells expressing high levels of TopBP1. We suggest that inhibition of rRNA transcription by different stress regulators is a general mechanism for cells to initiate nucleolar stress pathway., published version, http://purl.org/eprint/status/PeerReviewed

Hyaluronan synthases (HAS1–3) are unique in that they are active only when located in the plasma membrane, where they extrude the growing hyaluronan (HA) directly into cell surface and extracellular space. Therefore, traffic of HAS to/from the plasma membrane is crucial for the synthesis of HA. In this study, we have identified Rab10 GTPase as the first protein known to be involved in the control of this traffic. Rab10 colocalized with HAS3 in intracellular vesicular structures and was co-immunoprecipitated with HAS3 from isolated endosomal vesicles. Rab10 silencing increased the plasma membrane residence of HAS3, resulting in a significant increase of HA secretion and an enlarged cell surface HA coat, whereas Rab10 overexpression suppressed HA synthesis. Rab10 silencing blocked the retrograde traffic of HAS3 from the plasma membrane to early endosomes. The cell surface HA coat impaired cell adhesion to type I collagen, as indicated by recovery of adhesion following hyaluronidase treatment. The data indicate a novel function for Rab10 in reducing cell surface HAS3, suppressing HA synthesis, and facilitating cell adhesion to type I collagen. These are processes important in tissue injury, inflammation, and malignant growth.

Hyaluronan synthases (HAS) are unique plasma membrane glycosyltransferases secreting this glycosaminoglycan directly to the extracellular space. The three HAS isoenzymes (HAS1, HAS2, and HAS3) expressed in mammalian cells differ in their enzymatic properties and regulation by external stimuli, but clearly distinct functions have not been established. To overview the expression of different HAS isoenzymes during embryonic development and their subcellular localization, we immunostained mouse embryonic samples and cultured cells with HAS antibodies, correlating their distribution to hyaluronan staining. Their subcellular localization was further studied by GFP-HAS fusion proteins. Intense hyaluronan staining was observed throughout the development in the tissues of mesodermal origin, like heart and cartilages, but also for example during the maturation of kidneys and stratified epithelia. In general, staining for one or several HASs correlated with hyaluronan staining. The staining of HAS2 was most widespread, both spatially and temporally, correlating with hyaluronan staining especially in early mesenchymal tissues and heart. While epithelial cells were mostly negative for HASs, stratified epithelia became HAS positive during differentiation. All HAS isoenzymes showed cytoplasmic immunoreactivity, both in tissue sections and cultured cells, while plasma membrane staining was also detected, often in cellular extensions. HAS1 had brightest signal in Golgi, HAS3 in Golgi and microvillous protrusions, whereas most of the endogenous HAS2 immunoreactivity was localized in the ER. This differential pattern was also observed with transfected GFP-HASs. The large proportion of intracellular HASs suggests that HAS forms a reserve that is transported to the plasma membrane for rapid activation of hyaluronan synthesis.

Hyaluronan is a ubiquitous glycosaminoglycan involved in embryonic development, inflammation and cancer. In mammals, three hyaluronan synthase isoenzymes (HAS1-3) inserted in the plasma membrane produce hyaluronan directly on cell surface. The mRNA level and enzymatic activity of HAS1 are lower than those of HAS2 and HAS3 in many cells, obscuring the importance of HAS1. Here we demonstrate using immunocytochemistry and transfection of fluorescently tagged HAS1 that its enzymatic activity depends on the ER-Golgi-plasma membrane traffic, like reported for HAS2 and HAS3. When cultured in 5 mM glucose, HAS1-transfected MCF-7 cells show very little cell surface hyaluronan, detected with a fluorescent hyaluronan binding probe. However, a large hyaluronan coat was seen in cells grown in 20 mM glucose and 1 mM glucosamine, or treated with IL-1β, TNF-α, or TGF-β. The coats were mostly removed by the presence of hyaluronan hexasaccharides, or Hermes1 antibody, indicating that they depended on the CD44 receptor, which is in a contrast to the coat produced by HAS3, remaining attached to HAS3 itself. The findings suggest that HAS1-dependent coat is induced by inflammatory agents and glycemic stress, mediated by altered presentation of either CD44 or hyaluronan, and can offer a rapid cellular response to injury and inflammation.

Hyaluronan (HA), a natural extracellular matrix component, has been considered as an important constituent of the stem cell niche, and successfully used as 3D scaffolds for the chondrogenic differentiation of stem cells. However, the expression levels of HA synthases (HAS1, 2 and 3) and the synthesis of HA by stem cells have remained unknown, and were studied here in the human bone marrow-derived mesenchymal stem cells (hMSCs). Nine hMSCs from different donors were cultured as monolayers with MSC culture medium supplemented with FGF-2. The amount of HA secreted into medium was studied by an ELISA-type assay, and HA bound to cell surface by live cell microscopy. The expression of HASs was analyzed by real time RT-PCR and immunostainings. The HA receptor CD44 was studied by immunocytochemistry. An intense HA coat surrounded the plasma membrane and its protrusions in all nine hMSCs. Displacement assay with HA oligosaccharides indicated that HA coat was at least partly dependent on CD44, which showed similar, relatively high expression in all hMSCs. All HAS isoenzymes were detected, HAS1 showing the largest and HAS3 the smallest range of expression levels between the hMSCs. The secretion of HA ranged between 22.5 and 397.4 ng/10,000 cells/24h, and could not be clearly assigned to the mRNA level of a certain HAS, or a combination of the isoenzymes. This suggests that post-transcriptional and post-translational factors were involved in the adjustment of the HA secretion. In conclusion, all hMSCs expressed high levels of HAS1-3, secrete large amounts of HA, and surround themselves with a thick HA coat bound to CD44. The results suggest that hMSC has the potential for autocrine maintenance of the HA niche, important for their stemness.

Mammals have three homologous genes encoding proteins with hyaluronan synthase activity (Has1-3), all producing an identical polymer from UDP-N-acetylglucosamine and UDP-glucuronic acid. To compare the properties of these isoenzymes, COS-1 cells, with minor endogenous hyaluronan synthesis, were transfected with human Has1-3 isoenzymes. HAS1 was almost unable to secrete hyaluronan or form a hyaluronan coat, in contrast to HAS2 and HAS3. This failure of HAS1 to synthesize hyaluronan was compensated by increasing the cellular content of UDP-N-acetyl glucosamine by ∼10-fold with 1 mm glucosamine in the growth medium. Hyaluronan synthesis driven by HAS2 was less affected by glucosamine addition, and HAS3 was not affected at all. Glucose-free medium, leading to depletion of the UDP-sugars, markedly reduced hyaluronan synthesis by all HAS isoenzymes while raising its concentration from 5 to 25 mm had a moderate stimulatory effect. The results indicate that HAS1 is almost inactive in cells with low UDP-sugar supply, HAS2 activity increases with UDP-sugars, and HAS3 produces hyaluronan at high speed even with minimum substrate content. Transfected Has2 and particularly Has3 consumed enough UDP-sugars to reduce their content in COS-1 cells. Comparison of different human cell types revealed ∼50-fold differences in the content of UDP-N-acetylhexosamines and UDP-glucuronic acid, correlating with the expression level of Has1, suggesting cellular coordination between Has1 expression and the content of UDP-sugars.

Diffusely infiltrating astrocytomas are central nervous system tumors originating from astrocytic glial cells or their precursor cells. In this retrospective study we investigated the content of hyaluronan and the expression of hyaluronan metabolizing enzymes and its cell surface receptor, CD44, in these aggressive tumors. Hyaluronan is a large extracellular matrix molecule synthesized by three hyaluronan synthases (HAS1-3) on the plasma membrane and degraded by two hyaluronidases (HYAL1-2). In many tumors, aberrant hyaluronan metabolism implicates aggressive disease progression and metastatic potential. Our material consisted of 165 diffusely infiltrating astrocytomas (WHO grades II-IV). Tumor samples were processed into tissue microarray (TMA) blocks. The TMA sections were stained for hyaluronan, CD44, HAS1, HAS2, HAS3 and HYAL2. The staining intensity of astrocytomas was evaluated with a four-level scoring (0-3; no color, weak, moderate and strong) by two independent observers. The immunostaining results were compared with χ2 -test or with Kruskal-Wallis test for correlation with clinicopathological parameters and survival analyses were done with Kaplan-Meier log rank test and Cox regression. Hyaluronan and CD44 were strongly expressed in astrocytic gliomas but their expression did not correlate with WHO grade, cell proliferation activity by Ki-67, p53 status, epidermal growth factor receptor (EGFR) amplification or IDH1 mutation. Whereas HAS2 staining intensity showed a significant correlation with IDH1 mutation. Tumors with high HAS2 expression were IDH1 negative (p = 0.003). In addition, in non-parametric tests increased HAS2 staining intensity showed association with increased cell proliferation (p = 0.013) and in log rank test with decreased overall survival of patients (p = 0.001). Variables included in the multivariable Cox regression analysis were HAS2 staining intensity, p53 status, EGFR amplification, IDH1 mutation and WHO tumor grade; in this analysis HAS2 expression turned out to be a significant independent prognostic factor (p = 0.008). This study indicates that elevated expression of HAS2 is associated with glioma progression and suggests that HAS2 has a prognostic significance in diffusely infiltrating astrocytomas. Citation Format: Mari J. Poukka, Hannu Haapasalo, Kirsi J. Rilla, Kristiina Tyynelä-Korhonen, Ylermi Soini, Sanna M. Pasonen-Seppänen. Elevated expression of hyaluronan synthase 2 associates with poor prognosis in diffusely infiltrating astrocytomas. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3111.

The amount of hyaluronan (HA) is low in simple epithelia under normal conditions, but during tumorigenesis, trauma or inflammation HA is increased on the epithelial cells and surrounding stroma. Excessive HA in epithelia is suggested to interfere with cell–cell adhesions, resulting in disruption of the epithelial barrier function. In addition, stimulated HA synthesis has been correlated with epithelial-to-mesenchymal transition and invasion of cancer cells. However, the effects of HA overload on normal epithelial morphogenesis have not been characterized in detail. Madin-Darby canine kidney (MDCK) cells form polarized epithelial cysts, when grown in a 3-dimensional (3D) matrix. These cells were used to investigate whether stimulated HA synthesis, induced by stable overexpression of GFP-HAS3, influences cell polarization and epithelial morphogenesis. GFP-HAS3 expression in polarized MDCK cells resulted in active HA secretion at apical and basolateral membrane domains. HA-deposits interfered with the formation of cell–cell junctions, resulting in impaired barrier function. In 3D cyst cultures, HA accumulated into apical lumina and was also secreted from the basal side. The HAS3-expressing cysts failed to form a single lumen and instead displayed multiple small lumina. This phenotype was correlated with aberrant mitotic spindle orientation in dividing cells. The results of this study indicate that excess pericellular HA disturbs the normal cell–cell and cell–ECM interactions in simple epithelia, leading to aberrant epithelial morphogenesis. The morphological abnormalities observed in 3D epithelial cultures upon stimulated HAS3 expression may be related to premalignant changes, including intraluminal invasion and deregulated epithelialization, probably mediated by the mitotic spindle orientation defects.

Chronic intense UV radiation is the main cause of epidermal tumors. Because hyaluronan (HA), a large extracellular polysaccharide, is known to promote malignant growth, hyaluronan expression was studied in a model in which long-term UV radiation (UVR) induces epidermal tumors. Mouse back skin was exposed three times a week for 10.5 months to UVR corresponding to one minimal erythema dose, processed for histology, and stained for hyaluronan and the hyaluronan receptor CD44. This exposure protocol caused epidermal hyperplasia in most of the animals; tumors, mainly squamous cell carcinomas (SCCs), were found in ~20% of the animals. Specimens exposed to UVR showed increased hyaluronan and CD44 staining throughout the epidermal tissue. In hyperplastic areas, hyaluronan and CD44 stainings correlated positively with the degree of hyperplasia. Well-differentiated SCCs showed increased hyaluronan and CD44 staining intensities, whereas poorly differentiated tumors and dysplastic epidermis showed areas where HA and CD44 were locally reduced. The findings indicate that HA and CD44 increase in epidermal keratinocytes in the premalignant hyperplasia induced by UV irradiation and stay elevated in dysplasia and SCC, suggesting that the accumulation of hyaluronan and CD44 is an early marker for malignant transformation and may be a prerequisite for tumor formation.

Hyaluronan accumulation on cancer cells and their surrounding stroma predicts an unfavourable disease outcome, suggesting that hyaluronan enhances tumor growth and spreading. 4-Methylumbelliferone (4-MU) inhibits hyaluronan synthesis and retards cancer spreading in experimental animals through mechanisms not fully understood. These mechanisms were studied in A2058 melanoma cells, MCF-7 and MDA-MB-361 breast, SKOV-3 ovarian and UT-SCC118 squamous carcinoma cells by analysing hyaluronan synthesis, UDP-glucuronic acid (UDP-GlcUA) content, and hyaluronan synthase (HAS) mRNA levels. The maximal inhibition in hyaluronan synthesis ranged 22-80% in the cell lines tested. Active glucuronidation of 4-MU produced large quantities of 4-MU-glucuronide, depleting the cellular UDP-GlcUA pool. The maximal reduction varied between 38 and 95%. 4-MU also downregulated HAS mRNA levels: HAS3 was 84-60% lower in MDA-MB-361, A2058 and SKOV-3 cells. HAS2 was the major isoenzyme in MCF-7 cells and lowered by 81%, similar to 88% in A2058 cells. These data indicate that both HAS substrate and HAS2 and/or HAS3 mRNA are targeted by 4-MU. Despite different target point sensitivities, the reduction of hyaluronan caused by 4-MU was associated with a significant inhibition of cell migration, proliferation and invasion, supporting the importance of hyaluronan synthesis in cancer, and the therapeutic potential of hyaluronan synthesis inhibition.

The pathogenesis of age-related macular degeneration involves chronic oxidative stress, impaired degradation of membranous discs shed from photoreceptor outer segments and accumulation of lysosomal lipofuscin in retinal pigment epithelial (RPE) cells. It has been estimated that a major part of cellular proteolysis occurs in proteasomes, but the importance of proteasomes and the other proteolytic pathways including autophagy in RPE cells is poorly understood. Prior to proteolysis, heat shock proteins (Hsps), agents that function as molecular chaperones, attempt to refold misfolded proteins and thus prevent the accumulation of cytoplasmic protein aggregates. In the present study, the roles of the Hsp70 molecular chaperone and proteasomal and lysosomal proteolytic pathways were evaluated in human RPE cells (ARPE-19). The Hsp70 and ubiquitin protein levels and localization were analysed by Western blotting and immunofluorescense. Confocal and transmission electron microscopy were used to detect cellular organelles and to evaluate the morphological changes. Hsp70 levels were modulated using RNA interference and overexpression techniques. Cell viability was measured by colorimetric assay. The proteasome inhibitor MG-132 evoked the accumulation of perinuclear aggregates positive for Hsp70, ubiquitin-protein conjugates and the lysosomal membrane protein LAMP-2. Interestingly, the hsp70 mRNA depletion significantly increased cell death in conjunction with proteasome inhibition. We found that the accumulation of lysosomes was reversible: a cessation of proteasome inhibition led to clearance of the deposits via a mechanism believed to include autophagy. The molecular chaperone Hsp70, proteasomes and autophagy have an important regulatory role in the protein turnover of human RPE cells and may thus open new avenues for understanding degenerative processes in retinal cells.

Hyaluronan attached to cell surface can form at least two very different structures; a pericellular coat close to plasma membrane and hyaluronan chains coalesced into "cables" that can span several cell lengths. The hyaluronan in cables, induced by many inflammatory agents, can bind leukocytes, whereas that in the pericellular coat does not contribute to leukocyte binding. Therefore, this structural change seems to have a major role in inflammation. In the present study we checked whether cells of squamous epithelium, like epidermal keratinocytes, can form hyaluronan cables and bind leukocytes. In addition, we checked whether hyaluronan synthesis is affected during the induction of cables. Control keratinocytes expressed pericellular hyaluronan as small patches on plasma membrane. But when treated with inflammatory agents or stressful conditions (tunicamycin, interleukin-1beta, tumor necrosis factor-alpha, and high glucose concentration), hyaluronan organization changed into cable-like structures that avidly bound monocytes. Simultaneously, the total amount of secreted hyaluronan was slightly decreased, and the expression levels of hyaluronan synthases (Has1-3) and CD44 were not significantly changed. The results show that epidermal keratinocytes can form cables and bind leukocytes under inflammatory provocation and that these effects are not dependent on stimulation of hyaluronan secretion.

Hyaluronan synthases (HASs) are plasma membrane enzymes that simultaneously elongate, bind, and extrude the growing hyaluronan chain directly into extracellular space. In cells transfected with green fluorescent protein (GFP)-tagged Has3, the dorsal surface was decorated by up to 150 slender, 3-20-microm-long microvillus-type plasma membrane protrusions, which also contained filamentous actin, the hyaluronan receptor CD44, and lipid raft microdomains. Enzymatic activity of HAS was required for the growth of the microvilli, which were not present in cells transfected with other GFP proteins or inactive GFP-Has3 mutants or in cells incubated with exogenous soluble hyaluronan. The microvilli induced by HAS3 were gradually withered by introduction of an inhibitor of hyaluronan synthesis and rapidly retracted by hyaluronidase digestion, whereas they were not affected by competition with hyaluronan oligosaccharides and disruption of the CD44 gene, suggesting independence of hyaluronan receptors. The data bring out the novel concept that the glycocalyx created by dense arrays of hyaluronan chains, tethered to HAS during biosynthesis, can induce and maintain prominent microvilli.

Hyaluronan is a multifunctional glycosaminoglycan up to 10(7) Da molecular mass produced by the integral membrane glycosyltransferase, hyaluronan synthase (HAS). When expressed in keratinocytes, N-terminally tagged green fluorescent protein-HAS2 and -HAS3 isoenzymes were found to travel through endoplasmic reticulum (ER), Golgi, plasma membrane, and endocytic vesicles. A distinct enrichment of plasma membrane HAS was found in cell protrusions. The total turnover time of HAS3 was 4-5 h as judged by the green fluorescent protein signal decay and hyaluronan synthesis inhibition in cycloheximide-treated cells. The transfer from ER to Golgi took about 1 h, and the dwell time on the plasma membrane was less than 2 h in experiments with a relief and introduction, respectively, of brefeldin A. Constructs of HAS3 with 16- and 45-amino-acid C-terminal deletions mostly stayed within the ER, whereas a D216A missense mutant was localized within the Golgi complex but not the plasma membrane. Both types of mutations were almost or completely inactive, similar to the wild type enzyme that had its entry to the plasma membrane experimentally blocked by brefeldin A. Inhibition of hyaluronan synthesis by UDP-glucuronic acid starvation using 4-methyl-umbelliferone also prevented HAS access to the plasma membrane. The results demonstrate that 1) a latent pool of HAS exists within the ER-Golgi pathway; 2) this pool can be rapidly mobilized and activated by insertion into the plasma membrane; and 3) inhibition of HAS activity through mutation or substrate starvation results in exclusion of HAS from the plasma membrane.

Since excessive epidermal hyaluronan is associated with hyperproliferative states and disturbed terminal differentiation of the keratinocytes, we hypothesized that 4-methylumbelliferone (4-MU), an inhibitor of hyaluronan synthesis, could counteract these phenotypic features. Cultured epidermal keratinocytes showed a concentration dependent, maximum 83% reduction of hyaluronan in the presence of 0.2-1.0 mM 4-MU, whereas less decline was seen in the synthesis of chondroitin and heparan sulfate. The reduced hyaluronan was associated with no apparent change in its molecular mass. The 4-MU-treated keratinocytes showed an accentuated epithelial morphology with a flat, round cell shape, increased stress fibers and large vinculin-positive adhesion plaques, cytoskeletal changes consistent with the markedly reduced migration rate observed in scratched monolayer cultures. High concentrations of 4-MU also caused a block in keratinocyte proliferation, reversible upon 4-MU withdrawal. In the epidermis of organotypic cultures, 4-MU prevented the hyaluronan accumulation and epidermal hypertrophy induced by epidermal growth factor. The present results concur with earlier data indicating that enhanced cell locomotion and proliferation are associated with hyaluronan synthesis in activated keratinocytes. Cell proliferation, however, was blocked more strongly than expected on the basis of the incomplete hyaluronan synthesis inhibition, and may represent a novel target of 4-MU. At any rate, 4-MU and equivalent hyaluronan synthesis inhibitors might be considered for situations where suppression of epidermal activation and hyperproliferation is warranted.

Hyaluronan is a major component of the epidermal extracellular matrix, is actively synthesized by keratinocytes and shows fast matrix turnover in the stratified epithelium. We probed the importance of hyaluronan synthesis in keratinocytes by establishing cell lines carrying the exogenous hyaluronan synthase 2 (Has2) gene in sense and antisense orientations to increase and decrease their hyaluronan synthesis, respectively. Compared with cell lines transfected with the vector only, most clones containing the Has2 sense gene migrated faster in an in vitro wounding assay, whereas Has2 antisense cells migrated more slowly. Has2 antisense clones showed delayed entry into the S phase of cell cycle following plating, smaller lamellipodia and less spreading on the substratum. The decrease of hyaluronan on the undersurface of Has2 antisense cells was associated with an increased area of adhesion plaques containing vinculin. Exogenous hyaluronan added to the keratinocyte cultures had a minor stimulatory effect on migration after wounding but did not restore the reduced migratory ability of Has2 antisense cells. Hyaluronan decasaccharides that displace receptor bound hyaluronan in keratinocytes, and Streptomyces hyaluronidase sufficient to remove most cell surface hyaluronan had little effect on cell migration. The results suggest that the dynamic synthesis of hyaluronan directed by Has2, rather than the abundance of pericellular hyaluronan, controls keratinocyte migration, a cell function vital for the repair of squamous epithelia following wounding.

Hyaluronan (HA) is a linear polysaccharide abundant in the extracellular space between epidermal keratinocytes. It is synthesized at the inner face of the plasma membrane by hyaluronan synthases (Has). We probed the importance of hyaluronan in keratinocytes by establishing cell lines carrying exogenous hyaluronan synthase 2 (Has2) gene(s) in sense and antisense orientations in order to increase and decrease their hyaluronan synthesis, respectively. The cell lines with the sense Has2 cDNA showed increased HA synthesis, while most cell lines with Has2 antisense cDNA contained less HA. Has2 antisense cells differed from control cell lines; they spread at a slower rate and retained a rounded morphology for a longer time. Further, during the first 24 hours after plating, proliferation was delayed in the antisense cell lines. In an in vitro wounding assay the antisense cells showed a significantly decreased migration rate as compared to controls. Cell lines with the Has2 sense cDNA were similar to the control cell lines in spreading and proliferation rates. However, they migrated faster than control cell lines.

Rat epidermal keratinocyte monolayer cultures (REKs) actively synthesize hyaluronan (HA) most of which is retained on the cell surface or released into culture medium. However, a small proportion of HA also resides in an intracellular compartment (IC-HA). We characterized IC-HA localization and processing in REKs using specific staining with HA-binding probe, and its size by gel filtration of metabolically labeled HA. About 3% of REKs exhibited abundant IC-HA, while half of the cells lacked any microscopically demonstrable IC-HA. IC-HA was localized in 200–600 nm cytoplasmic vesicles. Dual staining of IC-HA with markers for lysosomes showed no colocalization. When REKs were treated with HA10 (decasaccharide), all IC-HA disappeared, while HA6, HA4, and sulfated glycosaminoglycans had no effect. All IC-HA was cleared 10 min after addition of HA10, and a 50%-reduction was reached in 5 min. An anti-CD44 mab, that increases HA on the cell surface, also increased IC-HA. Inhibiton of endocytosis via coated pits and caveolae did not reduce the amount of IC-HA. Perturbation of lysosomal activity caused accumulation of IC-HA. Most of the IC-HA had low MW ( 2000 kDa). The data demonstrate a rapid uptake and lysosomal degradation of IC-HA in REKs via a receptor dependent route separate from coated pits and caveolae, that involves a receptor with HA10 specificity, identified as CD44 or functionally dependent on CD44. The IC-HA consists of small HA fragments which may have a specific role in cellular homeostasis.

High concentrations of hyaluronan reside in the small space between the vital kertinocyte layers of human and animal epidermis and influence keratinocyte interactions, including growth, mobility and differentiation. We have previously found that the content of epidermal hyaluronan in human skin organ cultures is decreased and increased by cortisol and retinoic acid, and associated with enhanced and retarded terminal differentiation, respectively. To further substantiate this idea, we incubated epidermal keratinocytes with epidermal growth factor (EGF), and found a marked increase in hyaluronan synthesis which correlated with faster migration in an in vitro wounding assay of keratinocyte monolayers. EGF increased hyaluronan also in stratified, differentiated organotypic cultures, and increased the height of vital epidermis and reduced the thickness of the cornified layers, findings in line with an inhibition of terminal differentiation of keratinocytes. The stimulation of hyaluronan synthesis by EGF was due to upregulation of hyaluronan synthase 2 (HAS2) but not HAS1 or HAS3. A part of the EGF influence on the structure of epidermis, and on skin wound healing, is thus mediated through its control of HAS2 expression.

Hyaluronan synthesized in the epidermis has an exceptionally short half-life, indicative of its catabolism by epidermal keratinocytes. An intracellular pool of endogenously synthesized hyaluronan, from 1 to 20 fg/cell, inversely related to cell density, was observed in cultured rat epidermal keratinocytes. More than 80% of the intracellular hyaluronan was small (