International Journal of Cell Biology The latest articles from Hindawi © 2018 , Hindawi Limited . All rights reserved. Nesprin-2 Interacts with Condensin Component SMC2 Wed, 27 Dec 2017 00:00:00 +0000 The nuclear envelope proteins, Nesprins, have been primarily studied during interphase where they function in maintaining nuclear shape, size, and positioning. We analyze here the function of Nesprin-2 in chromatin interactions in interphase and dividing cells. We characterize a region in the rod domain of Nesprin-2 that is predicted as SMC domain (aa 1436–1766). We show that this domain can interact with itself. It furthermore has the capacity to bind to SMC2 and SMC4, the core subunits of condensin. The interaction was observed during all phases of the cell cycle; it was particularly strong during S phase and persisted also during mitosis. Nesprin-2 knockdown did not affect condensin distribution; however we noticed significantly higher numbers of chromatin bridges in Nesprin-2 knockdown cells in anaphase. Thus, Nesprin-2 may have an impact on chromosomes which might be due to its interaction with condensins or to indirect mechanisms provided by its interactions at the nuclear envelope. Xin Xing, Carmen Mroß, Linlin Hao, Martina Munck, Alexandra Herzog, Clara Mohr, C. P. Unnikannan, Pranav Kelkar, Angelika A. Noegel, Ludwig Eichinger, and Sascha Neumann Copyright © 2017 Xin Xing et al. All rights reserved. Thrombin-Induced Calpain Activation Promotes Protease-Activated Receptor 1 Internalization Thu, 09 Nov 2017 06:51:50 +0000 The serine protease thrombin activates Protease-Activated Receptors (PARs), a family of G-protein-coupled receptors (GPCRs) activated by the proteolytic cleavage of their extracellular N-terminal domain. Four members of this family have been identified: PAR1–4. The activation of Protease-Activated Receptor 1(PAR1), the prototype of this receptor family, leads to an increase in intracellular Ca+2 concentration ([Ca+2]i) mediated by α coupling and phospholipase C (PLC) activation. We have previously shown that the stimulation of PAR1 by thrombin promotes intracellular signaling leading to RPE cell transformation, proliferation, and migration which characterize fibroproliferative eye diseases leading to blindness. Within this context, the elucidation of the mechanisms involved in PAR1 inactivation is of utmost importance. Due to the irreversible nature of PAR1 activation, its inactivation must be efficiently regulated in order to terminate signaling. Using ARPE-19 human RPE cell line, we characterized thrombin-induced [Ca+2]i increase and demonstrated the calcium-dependent activation of μ-calpain mediated by PAR1. Calpains are a family of calcium-activated cysteine proteases involved in multiple cellular processes including the internalization of membrane proteins through clathrin-coated vesicles. We demonstrated that PAR1-induced calpain activation results in the degradation of α-spectrin by calpain, essential for receptor endocytosis, and the consequent decrease in PAR1 membrane expression. Collectively, the present results identify a novel μ-calpain-dependent mechanism for PAR1 inactivation following exposure to thrombin. Alejandro Alvarez-Arce, Irene Lee-Rivera, Edith López, Arturo Hernández-Cruz, and Ana María López-Colomé Copyright © 2017 Alejandro Alvarez-Arce et al. All rights reserved. Effect of Antioxidant Water on the Bioactivities of Cells Thu, 17 Aug 2017 09:11:54 +0000 It has been reported that water at the interface of a hydrophilic thin film forms an exclusion zone, which has a higher density than ordinary water. A similar phenomenon was observed for a hydrated hydrophilic ceramic powder, and water turns into a three-dimensional cell-like structure composed of high density water and low density water. This structured water appears to have a stimulative effect on plant growth. This report outlines our study of antioxidant properties of this structured water and its effect on cell bioactivities. Culturing media which were prepared utilizing this antioxidant structured water promoted the viability of RAW 264.7 macrophage cells by up to three times. The same tendency was observed for other cells including IEC-6, C2C12, and 3T3-L1. Also, the cytokine expression of the splenocytes taken from a mouse spleen increased in the same manner. The water also appears to suppress the viability of cancer cell, MCF-7. These results strongly suggest that the structured water helps the activities of normal cells while suppressing those of malignant cells. Seong Gu Hwang, Ho-Sung Lee, Byung-Cheon Lee, and GunWoong Bahng Copyright © 2017 Seong Gu Hwang et al. All rights reserved. Regeneration and Regrowth Potentials of Digit Tips in Amphibians and Mammals Mon, 10 Apr 2017 00:00:00 +0000 Tissue regeneration and repair have received much attention in the medical field over the years. The study of amphibians, such as newts and salamanders, has uncovered many of the processes that occur in these animals during full-limb/digit regeneration, a process that is highly limited in mammals. Understanding these processes in amphibians could shed light on how to develop and improve this process in mammals. Amputation injuries in mammals usually result in the formation of scar tissue with limited regrowth of the limb/digit; however, it has been observed that the very tips of digits (fingers and toes) can partially regrow in humans and mice under certain conditions. This review will summarize and compare the processes involved in salamander limb regeneration, mammalian wound healing, and digit regeneration in mice and humans. Yohan Choi, Fanwei Meng, Charles S. Cox, Kevin P. Lally, Johnny Huard, and Yong Li Copyright © 2017 Yohan Choi et al. All rights reserved. Thrombopoietin Secretion by Human Ovarian Cancer Cells Thu, 30 Mar 2017 12:30:03 +0000 The thrombopoietin (TPO) gene expression in human ovary and cancer cells from patients with ovarian carcinomatosis, as well as several cancer cell lines including MDA-MB231 (breast cancer), K562 and HL60 (Leukemic cells), OVCAR-3NIH and SKOV-3 (ovarian cancer), was performed using RT PCR, real-time PCR, and gene sequencing. Human liver tissues are used as controls. The presence of TPO in the cells and its regulation by activated protein C were explored by flow cytometry. TPO content of cell extract as well as plasma of a patient with ovarian cancer was evaluated by ELISA. The functionality of TPO was performed in coculture on the basis of the viability of a TPO-dependent cell line (Ba/F3), MTT assay, and Annexin-V labeling. As in liver, ovarian tissues and all cancer cells lines except the MDA-MB231 express the three TPO-1 (full length TPO), TPO-2 (12 bp deletion), and TPO-3 (116 pb deletion) variants. Primary ovarian cancer cells as well as cancer cell lines produce TPO. The thrombopoietin production by OVCAR-3 increased when cells are stimulated by aPC. OVCAR-3 cell’s supernatant can replace exogenous TPO and inhibited TPO-dependent cell line (Ba/F3) apoptosis. The thrombopoietin produced by tumor may have a direct effect on thrombocytosis/thrombosis occurrence in patients with ovarian cancer. Samaher Besbes, Shahid Shah, Iman Al-dybiat, Shahsoltan Mirshahi, Helene Helfer, Haythem Najah, Caroline Fourgeaud, Marc Pocard, Ibtissem Ghedira, Jeannette Soria, and Massoud Mirshahi Copyright © 2017 Samaher Besbes et al. All rights reserved. Class-Specific Histone Deacetylase Inhibitors Promote 11-Beta Hydroxysteroid Dehydrogenase Type 2 Expression in JEG-3 Cells Tue, 21 Feb 2017 00:00:00 +0000 Exposure to maternal cortisol plays a crucial role in fetal organogenesis. However, fetal overexposure to cortisol has been linked to a range of short- and long-term adverse outcomes. Normally, this is prevented by the expression of an enzyme in the placenta called 11-beta hydroxysteroid dehydrogenase type 2 (11β-HSD2) which converts active cortisol to its inactive metabolite cortisone. Placental 11β-HSD2 is known to be reduced in a number of adverse pregnancy complications, possibly through an epigenetic mechanism. As a result, a number of pan-HDAC inhibitors have been examined for their ability to promote 11β-HSD2 expression. However, it is not known if the effects of pan-HDAC inhibition are a general phenomenon or if the effects are dependent upon a specific class of HDACs. Here, we examined the ability of pan- and class-specific HDAC inhibitors to regulate 11β-HSD2 expression in JEG3 cells. We find that pan-, class I, or class IIa HDAC inhibition promoted 11β-HSD2 expression and prevented cortisol or interleukin-1β-induced decrease in its expression. These results demonstrate that targeting a specific class of HDACs can promote 11β-HSD2 expression in JEG3 cells. This adds to the growing body of evidence suggesting that HDACs may be crucial in maintaining normal fetal development. Katie L. Togher, Louise C. Kenny, and Gerard W. O’Keeffe Copyright © 2017 Katie L. Togher et al. All rights reserved. Pulling a Ligase out of a “HAT”: pCAF Mediates Ubiquitination of the Class II Transactivator Sun, 12 Feb 2017 00:00:00 +0000 The Class II Transactivator (CIITA) is essential to the regulation of Major Histocompatibility Class II (MHC II) genes transcription. As the “master regulator” of MHC II transcription, CIITA regulation is imperative and requires various posttranslational modifications (PTMs) in order to facilitate its role. Previously we identified various ubiquitination events on CIITA. Monoubiquitination is important for CIITA transactivity, while K63 linked ubiquitination is involved in crosstalk with ERK1/2 phosphorylation, where together they mediate cellular movement from the cytoplasm to nuclear region. Further, CIITA is also modified by degradative K48 polyubiquitination. However, the E3 ligase responsible for these modifications was unknown. We show CIITA ubiquitination and transactivity are enhanced with the histone acetyltransferase (HAT), p300/CBP associated factor (pCAF), and the E3 ligase region within pCAF is necessary for both. Additionally, pCAF mediated ubiquitination is independent of pCAF’s HAT domain, and acetylation deficient CIITA is K48 polyubiquitinated and degraded in the presence of pCAF. Lastly, we identify the histone acetyltransferase, pCAF, as the E3 ligase responsible for CIITA’s ubiquitination. Julie E. Morgan and Susanna F. Greer Copyright © 2017 Julie E. Morgan and Susanna F. Greer. All rights reserved. Biologics for Targeting Inflammatory Cytokines, Clinical Uses, and Limitations Mon, 19 Dec 2016 11:09:45 +0000 Proinflammatory cytokines are potent mediators of numerous biological processes and are tightly regulated in the body. Chronic uncontrolled levels of such cytokines can initiate and derive many pathologies, including incidences of autoimmunity and cancer. Therefore, therapies that regulate the activity of inflammatory cytokines, either by supplementation of anti-inflammatory recombinant cytokines or by neutralizing them by using blocking antibodies, have been extensively used over the past decades. Over the past few years, new innovative biological agents for blocking and regulating cytokine activities have emerged. Here, we review some of the most recent approaches of cytokine targeting, focusing on anti-TNF antibodies or recombinant TNF decoy receptor, recombinant IL-1 receptor antagonist (IL-1Ra) and anti-IL-1 antibodies, anti-IL-6 receptor antibodies, and TH17 targeting antibodies. We discuss their effects as biologic drugs, as evaluated in numerous clinical trials, and highlight their therapeutic potential as well as emphasize their inherent limitations and clinical risks. We suggest that while systemic blocking of proinflammatory cytokines using biological agents can ameliorate disease pathogenesis and progression, it may also abrogate the hosts defense against infections. Moreover, we outline the rational need to develop new therapies, which block inflammatory cytokines only at sites of inflammation, while enabling their function systemically. Peleg Rider, Yaron Carmi, and Idan Cohen Copyright © 2016 Peleg Rider et al. All rights reserved. Localisation of Lactate Transporters in Rat and Rabbit Placentae Sun, 23 Oct 2016 13:28:09 +0000 The distribution of monocarboxylate transporter (MCT) isoforms 1 and 4, which mediate the plasmalemmal transport of L-lactic and pyruvic acids, has been identified in the placentae of rats and rabbits at different ages of gestation. Groups of three pregnant Sprague-Dawley rats and New Zealand White rabbits were sacrificed on gestation days (GD) 11, 14, 18, or 20 and on GD 13, 18, or 28, respectively. Placentae were removed and processed for immunohistochemical detection of MCT1 and MCT4. In the rat, staining for MCT1 was associated with lakes and blood vessels containing enucleated red blood cells (maternal vessels) while staining for MCT4 was associated with vessels containing nucleated red blood cells (embryofoetal vessels). In the rabbit, staining for MCT1 was associated with blood vessels containing nucleated red blood cells while staining for MCT4 was associated with vessels containing enucleated red blood cells. Strength of staining for MCT1 decreased during gestation in both species, but that for MCT4 was stronger than that for MCT1 and was consistent between gestation days. The results imply an opposite polarity of MCT1 and MCT4 across the trophoblast between rat and rabbit. Nigel P. Moore, Catherine A. Picut, and Jeffrey H. Charlap Copyright © 2016 Nigel P. Moore et al. All rights reserved. Microwave-Assisted Tissue Preparation for Rapid Fixation, Decalcification, Antigen Retrieval, Cryosectioning, and Immunostaining Thu, 20 Oct 2016 11:10:42 +0000 Microwave irradiation of tissue during fixation and subsequent histochemical staining procedures significantly reduces the time required for incubation in fixation and staining solutions. Minimizing the incubation time in fixative reduces disruption of tissue morphology, and reducing the incubation time in staining solution or antibody solution decreases nonspecific labeling. Reduction of incubation time in staining solution also decreases the level of background noise. Microwave-assisted tissue preparation is applicable for tissue fixation, decalcification of bone tissues, treatment of adipose tissues, antigen retrieval, and other special staining of tissues. Microwave-assisted tissue fixation and staining are useful tools for histological analyses. This review describes the protocols using microwave irradiation for several essential procedures in histochemical studies, and these techniques are applicable to other protocols for tissue fixation and immunostaining in the field of cell biology. Kazuo Katoh Copyright © 2016 Kazuo Katoh. All rights reserved. Carbohydrate Moieties and Cytoenzymatic Characterization of Hemocytes in Whiteleg Shrimp Litopenaeus vannamei Wed, 19 Oct 2016 11:09:01 +0000 Hemocytes represent one of the most important defense mechanisms against foreign material in Crustacea and are also involved in a variety of other physiological responses. Fluorescent lectin-binding assays and cytochemical reactions were used to identify specificity and distribution of carbohydrate moieties and presence of several hydrolytic enzymes, in hemocytes of whiteleg shrimp Litopenaeus vannamei. Two general classes of circulating hemocytes (granular and agranular) exist in L. vannamei, which express carbohydrates residues for FITC-conjugated lectins WGA, LEA, and PNA; UEA and Con-A were not observed. Enzymatic studies indicated that acid phosphatase, nonspecific esterase, and specific esterases were present; alkaline phosphatase was not observed. The enzymes and carbohydrates are useful tools in hemocyte classification and cellular defense mechanism studies. Norma Estrada, Edwin Velázquez, Carmen Rodríguez-Jaramillo, and Felipe Ascencio Copyright © 2016 Norma Estrada et al. All rights reserved. Connexin’s Connection in Breast Cancer Growth and Progression Tue, 23 Aug 2016 13:18:40 +0000 Gap junctions are cell-to-cell junctions that are located in the basolateral surface of two adjoining cells. A gap junction channel is composed of a family of proteins called connexins. Gap junction channels maintain intercellular communication between two cells through the exchange of ions, small metabolites, and electrical signals. Gap junction channels or connexins are widespread in terms of their expression and function in maintaining the development, differentiation, and homeostasis of vertebrate tissues. Gap junction connexins play a major role in maintaining intercellular communication among different cell types of normal mammary gland for proper development and homeostasis. Connexins have also been implicated in the pathogenesis of breast cancer. Differential expression pattern of connexins and their gap junction dependent or independent functions provide pivotal cross talk of breast tumor cells with the surrounding stromal cell in the microenvironment. Substantial research from the last 20 years has accumulated ample evidences that allow us a better understanding of the roles that connexins play in the tumorigenesis of primary breast tumor and its metastatic progression. This review will summarize the knowledge about the connexins and gap junction activities in breast cancer highlighting the differential expression and functional dynamics of connexins in the pathogenesis of the disease. Debarshi Banerjee Copyright © 2016 Debarshi Banerjee. All rights reserved. Stem Cells Applications in Regenerative Medicine and Disease Therapeutics Tue, 19 Jul 2016 12:53:11 +0000 Regenerative medicine, the most recent and emerging branch of medical science, deals with functional restoration of tissues or organs for the patient suffering from severe injuries or chronic disease. The spectacular progress in the field of stem cell research has laid the foundation for cell based therapies of disease which cannot be cured by conventional medicines. The indefinite self-renewal and potential to differentiate into other types of cells represent stem cells as frontiers of regenerative medicine. The transdifferentiating potential of stem cells varies with source and according to that regenerative applications also change. Advancements in gene editing and tissue engineering technology have endorsed the ex vivo remodelling of stem cells grown into 3D organoids and tissue structures for personalized applications. This review outlines the most recent advancement in transplantation and tissue engineering technologies of ESCs, TSPSCs, MSCs, UCSCs, BMSCs, and iPSCs in regenerative medicine. Additionally, this review also discusses stem cells regenerative application in wildlife conservation. Ranjeet Singh Mahla Copyright © 2016 Ranjeet Singh Mahla. All rights reserved. Measured Effects of Wnt3a on Proliferation of HEK293T Cells Depend on the Applied Assay Tue, 22 Dec 2015 11:12:14 +0000 The Wnt signaling pathway has been associated with many essential cell processes. This study aims to examine the effects of Wnt signaling on proliferation of cultured HEK293T cells. Cells were incubated with Wnt3a, and the activation of the Wnt pathway was followed by analysis of the level of the β-catenin protein and of the expression levels of the target genes MYC and CCND1. The level of β-catenin protein increased up to fourfold. While the mRNA levels of c-Myc and cyclin D1 increased slightly, the protein levels increased up to a factor of 1.5. Remarkably, MTT and BrdU assays showed different results when measuring the proliferation rate of Wnt3a stimulated HEK293T cells. In the BrdU assays an increase of the proliferation rate could be detected, which correlated to the applied Wnt3a concentration. Oppositely, this correlation could not be shown in the MTT assays. The MTT results, which are based on the mitochondrial activity, were confirmed by analysis of the succinate dehydrogenase complex by immunofluorescence and by western blotting. Taken together, our study shows that Wnt3a activates proliferation of HEK293 cells. These effects can be detected by measuring DNA synthesis rather than by measuring changes of mitochondrial activity. Patricia Reischmann, Johanna Fiebeck, Nadine von der Weiden, and Oliver Müller Copyright © 2015 Patricia Reischmann et al. All rights reserved. Integration of Mitochondrial Targeting for Molecular Cancer Therapeutics Wed, 02 Dec 2015 11:24:55 +0000 Mitochondrial metabolism greatly influences cancer cell survival, invasion, metastasis, and resistance to many anticancer drugs. Furthermore, molecular-targeted therapies (e.g., oncogenic kinase inhibitors) create a dependence of surviving cells on mitochondrial metabolism. For these reasons, inhibition of mitochondrial metabolism represents promising therapeutic pathways in cancer. This review provides an overview of mitochondrial metabolism in cancer and discusses the limitations of mitochondrial inhibition for cancer treatment. Finally, we present preclinical evidence that mitochondrial inhibition could be associated with oncogenic “drivers” inhibitors, which may lead to innovative drug combinations for improving the efficacy of molecular-targeted therapy. Philippe Marchetti, Pierre Guerreschi, Laurent Mortier, and Jerome Kluza Copyright © 2015 Philippe Marchetti et al. All rights reserved. Effects of Activating Mutations on EGFR Cellular Protein Turnover and Amino Acid Recycling Determined Using SILAC Mass Spectrometry Tue, 24 Nov 2015 11:56:06 +0000 Rapid mutations of proteins that are targeted in cancer therapy often lead to drug resistance. Often, the mutation directly affects a drug’s binding site, effectively blocking binding of the drug, but these mutations can have other effects such as changing the protein turnover half-life. Utilizing SILAC MS, we measured the cellular turnover rates of an important non-small cell lung cancer target, epidermal growth factor receptor (EGFR). Wild-type (WT) EGFR, EGFR with a single activating mutant (Del 746–750 or L858R), and the drug-resistant double mutant (L858R/T790M) EGFR were analyzed. In non-small cell lung cancer cell lines, EGFR turnover rates ranged from 28 hours in A431 cells (WT) to 7.5 hours in the PC-9 cells (Del 746–750 mutant). The measurement of EGFR turnover rate in PC-9 cells dosed with irreversible inhibitors has additional complexity due to inhibitor effects on cell viability and results were reported as a range. Finally, essential amino acid recycling (K and R) was measured in different cell lines. The recycling was different in each cell line, but the overall inclusion of the effect of amino acid recycling on calculating EGFR turnover rates resulted in a 10–20% reduction in rates. Michael J. Greig, Sherry Niessen, Scott L. Weinrich, Jun Li Feng, Manli Shi, and Ted O. Johnson Copyright © 2015 Michael J. Greig et al. All rights reserved. A Simple and Efficient Method for Preparing Cell Slides and Staining without Using Cytocentrifuge and Cytoclips Tue, 17 Nov 2015 11:17:14 +0000 Cell staining is a necessary and useful technique for visualizing cell morphology and structure under a microscope. This technique has been used in many areas such as cytology, hematology, oncology, histology, virology, serology, microbiology, cell biology, and immunochemistry. One of the key pieces of equipment for preparing a slide for cell staining is cytology centrifuge (cytocentrifuge) such as cytospin. However, many small labs do not have this expensive equipment and its accessory, cytoclips (also expensive relatively), which makes them difficult to study cell cytology. Here we present an alternative method for preparing a slide and cell staining in the absence of a cytocentrifuge (and cytoclips). This method is based on the principle that a regular cell centrifuge can be used to concentrate cells harvested from cell culture and then deposit the concentrated cell suspension to a slide evenly by using a cell spreader, followed by cell staining. The method presented is simple, rapid, economic, and efficient. This method may also avoid a possible change in cell morphology induced by cytocentrifuge. Xiaotang Hu, Verronika Laguerre, Daniel Packert, Alice Nakasone, and Lynn Moscinski Copyright © 2015 Xiaotang Hu et al. All rights reserved. Advances in Hyaluronan Biology: Signaling, Regulation, and Disease Mechanisms Sun, 13 Sep 2015 05:56:06 +0000 Melanie A. Simpson, Carol de la Motte, Larry S. Sherman, and Paul H. Weigel Copyright © 2015 Melanie A. Simpson et al. All rights reserved. Regulatory T Cells Resist Cyclosporine-Induced Cell Death via CD44-Mediated Signaling Pathways Thu, 10 Sep 2015 13:31:48 +0000 Cyclosporine A (CSA) is an immunosuppressive agent that specifically targets T cells and also increases the percentage of pro-tolerogenic CD4+Foxp3+ regulatory T cells (Treg) through unknown mechanisms. We previously reported that CD44, a receptor for the extracellular matrix glycosaminoglycan hyaluronan (HA), promotes Treg stability in IL-2-low environments. Here, we asked whether CD44 signaling also promotes Treg resistance to CSA. We found that CD44 cross-linking promoted Foxp3 expression and Treg viability in the setting of CSA treatment. This effect was IL-2 independent but could be suppressed using sc-355979, an inhibitor of Stat5-phosphorylation. Moreover, we found that inhibition of HA synthesis impairs Treg homeostasis but that this effect could be overcome with exogenous IL-2 or CD44-cross-linking. Together, these data support a model whereby CD44 cross-linking by HA promotes IL-2-independent Foxp3 expression and Treg survival in the face of CSA. Shannon M. Ruppert, Ben A. Falk, S. Alice Long, and Paul L. Bollyky Copyright © 2015 Shannon M. Ruppert et al. All rights reserved. High Sensitivity Method to Estimate Distribution of Hyaluronan Molecular Sizes in Small Biological Samples Using Gas-Phase Electrophoretic Mobility Molecular Analysis Thu, 10 Sep 2015 13:22:48 +0000 Hyaluronan is a negatively charged polydisperse polysaccharide where both its size and tissue concentration play an important role in many physiological and pathological processes. The various functions of hyaluronan depend on its molecular size. Up to now, it has been difficult to study the role of hyaluronan in diseases with pathological changes in the extracellular matrix where availability is low or tissue samples are small. Difficulty to obtain large enough biopsies from human diseased tissue or tissue from animal models has also restricted the study of hyaluronan. In this paper, we demonstrate that gas-phase electrophoretic molecular mobility analyzer (GEMMA) can be used to estimate the distribution of hyaluronan molecular sizes in biological samples with a limited amount of hyaluronan. The low detection level of the GEMMA method allows for estimation of hyaluronan molecular sizes from different parts of small organs. Hence, the GEMMA method opens opportunity to attain a profile over the distribution of hyaluronan molecular sizes and estimate changes caused by disease or experimental conditions that has not been possible to obtain before. Lan Do, Christen P. Dahl, Susanne Kerje, Peter Hansell, Stellan Mörner, Ulla Lindqvist, Anna Engström-Laurent, Göran Larsson, and Urban Hellman Copyright © 2015 Lan Do et al. All rights reserved. Correlative Light and Electron Microscopy Reveals the HAS3-Induced Dorsal Plasma Membrane Ruffles Thu, 10 Sep 2015 13:19:33 +0000 Hyaluronan is a linear sugar polymer synthesized by three isoforms of hyaluronan synthases (HAS1, 2, and 3) that forms a hydrated scaffold around cells and is an essential component of the extracellular matrix. The morphological changes of cells induced by active hyaluronan synthesis are well recognized but not studied in detail with high resolution before. We have previously found that overexpression of HAS3 induces growth of long plasma membrane protrusions that act as platforms for hyaluronan synthesis. The study of these thin and fragile protrusions is challenging, and they are difficult to preserve by fixation unless they are adherent to the substrate. Thus their structure and regulation are still partly unclear despite careful imaging with different microscopic methods in several cell types. In this study, correlative light and electron microscopy (CLEM) was utilized to correlate the GFP-HAS3 signal and the surface ultrastructure of cells in order to study in detail the morphological changes induced by HAS3 overexpression. Surprisingly, this method revealed that GFP-HAS3 not only localizes to ruffles but in fact induces dorsal ruffle formation. Dorsal ruffles regulate diverse cellular functions, such as motility, regulation of glucose metabolism, spreading, adhesion, and matrix degradation, the same functions driven by active hyaluronan synthesis. Kirsi Rilla and Arto Koistinen Copyright © 2015 Kirsi Rilla and Arto Koistinen. All rights reserved. Regulated Hyaluronan Synthesis by Vascular Cells Thu, 10 Sep 2015 13:18:33 +0000 Cellular microenvironment plays a critical role in several pathologies including atherosclerosis. Hyaluronan (HA) content often reflects the progression of this disease in promoting vessel thickening and cell migration. HA synthesis is regulated by several factors, including the phosphorylation of HA synthase 2 (HAS2) and other covalent modifications including ubiquitination and O-GlcNAcylation. Substrate availability is important in HA synthesis control. Specific drugs reducing the UDP precursors are able to reduce HA synthesis whereas the hexosamine biosynthetic pathway (HBP) increases the concentration of HA precursor UDP-N-acetylglucosamine (UDP-GlcNAc) leading to an increase of HA synthesis. The flux through the HBP in the regulation of HA biosynthesis in human aortic vascular smooth muscle cells (VSMCs) was reported as a critical aspect. In fact, inhibiting O-GlcNAcylation reduced HA production whereas increased O-GlcNAcylation augmented HA secretion. Additionally, O-GlcNAcylation regulates HAS2 gene expression resulting in accumulation of its mRNA after induction of O-GlcNAcylation with glucosamine treatments. The oxidized LDLs, the most common molecules related to atherosclerosis outcome and progression, are also able to induce a strong HA synthesis when they are in contact with vascular cells. In this review, we present recent described mechanisms involved in HA synthesis regulation and their role in atherosclerosis outcome and development. Manuela Viola, Evgenia Karousou, Maria Luisa D’Angelo, Ilaria Caon, Giancarlo De Luca, Alberto Passi, and Davide Vigetti Copyright © 2015 Manuela Viola et al. All rights reserved. Extracellular Vesicles from Caveolin-Enriched Microdomains Regulate Hyaluronan-Mediated Sustained Vascular Integrity Thu, 10 Sep 2015 13:06:28 +0000 Defects in vascular integrity are an initiating factor in several disease processes. We have previously reported that high molecular weight hyaluronan (HMW-HA), a major glycosaminoglycan in the body, promotes rapid signal transduction in human pulmonary microvascular endothelial cells (HPMVEC) leading to barrier enhancement. In contrast, low molecular weight hyaluronan (LMW-HA), produced in disease states by hyaluronidases and reactive oxygen species (ROS), induces HPMVEC barrier disruption. However, the mechanism(s) of sustained barrier regulation by HA are poorly defined. Our results indicate that long-term (6–24 hours) exposure of HMW-HA induced release of a novel type of extracellular vesicle from HLMVEC called enlargeosomes (characterized by AHNAK expression) while LMW-HA long-term exposure promoted release of exosomes (characterized by CD9, CD63, and CD81 expression). These effects were blocked by inhibiting caveolin-enriched microdomain (CEM) formation. Further, inhibiting enlargeosome release by annexin II siRNA attenuated the sustained barrier enhancing effects of HMW-HA. Finally, exposure of isolated enlargeosomes to HPMVEC monolayers generated barrier enhancement while exosomes led to barrier disruption. Taken together, these results suggest that differential release of extracellular vesicles from CEM modulate the sustained HPMVEC barrier regulation by HMW-HA and LMW-HA. HMW-HA-induced specialized enlargeosomes can be a potential therapeutic strategy for diseases involving impaired vascular integrity. Tamara Mirzapoiazova, Frances E. Lennon, Bolot Mambetsariev, Michael Allen, Jacob Riehm, Valeriy A. Poroyko, and Patrick A. Singleton Copyright © 2015 Tamara Mirzapoiazova et al. All rights reserved. The Rise and Fall of Hyaluronan in Respiratory Diseases Thu, 10 Sep 2015 12:21:45 +0000 In normal airways, hyaluronan (HA) matrices are primarily located within the airway submucosa, pulmonary vasculature walls, and, to a lesser extent, the alveoli. Following pulmonary injury, elevated levels of HA matrices accumulate in these regions, and in respiratory secretions, correlating with the extent of injury. Animal models have provided important insight into the role of HA in the onset of pulmonary injury and repair, generally indicating that the induction of HA synthesis is an early event typically preceding fibrosis. The HA that accumulates in inflamed airways is of a high molecular weight (>1600 kDa) but can be broken down into smaller fragments (<150 kDa) by inflammatory and disease-related mechanisms that have profound effects on HA pathobiology. During inflammation in the airways, HA is often covalently modified with heavy chains from inter-alpha-inhibitor via the enzyme tumor-necrosis-factor-stimulated-gene-6 (TSG-6) and this modification promotes the interaction of leukocytes with HA matrices at sites of inflammation. The clearance of HA and its return to normal levels is essential for the proper resolution of inflammation. These data portray HA matrices as an important component of normal airway physiology and illustrate its integral roles during tissue injury and repair among a variety of respiratory diseases. Mark E. Lauer, Raed A. Dweik, Stavros Garantziotis, and Mark A. Aronica Copyright © 2015 Mark E. Lauer et al. All rights reserved. Size Matters: Molecular Weight Specificity of Hyaluronan Effects in Cell Biology Thu, 10 Sep 2015 12:18:21 +0000 Hyaluronan signaling properties are unique among other biologically active molecules, that they are apparently not influenced by postsynthetic molecular modification, but by hyaluronan fragment size. This review summarizes the current knowledge about the generation of hyaluronan fragments of different size and size-dependent differences in hyaluronan signaling as well as their downstream biological effects. Jaime M. Cyphert, Carol S. Trempus, and Stavros Garantziotis Copyright © 2015 Jaime M. Cyphert et al. All rights reserved. Hyaluronan Synthase 3 Null Mice Exhibit Decreased Intestinal Inflammation and Tissue Damage in the DSS-Induced Colitis Model Thu, 10 Sep 2015 12:12:26 +0000 Hyaluronan (HA) overproduction is a hallmark of multiple inflammatory diseases, including inflammatory bowel disease (IBD). Hyaluronan can act as a leukocyte recruitment molecule and in the most common mouse model of intestinal inflammation, the chemically induced dextran sodium sulfate (DSS) experimental colitis model, we previously determined that changes in colon distribution of HA occur before inflammation. Therefore, we hypothesized that, during a pathologic challenge, HA promotes inflammation. In this study, we tested the progression of inflammation in mice null for the hyaluronan synthase genes (HAS1, HAS3, or both HAS1 and HAS3) in the DSS-colitis model. Our data demonstrate that both the HAS1/HAS3 double and the HAS3 null mice are protected from colitis, compared to wild-type and HAS1 null mice, as determined by measurement of weight loss, disease activity, serum IL-6 levels, histologic scoring, and immunohistochemistry. Most notable is the dramatic increase in submucosal microvasculature, hyaluronan deposition, and leukocyte infiltration in the inflamed colon tissue of wild-type and HAS1 null mice. Our data suggest, HAS3 plays a crucial role in driving gut inflammation. Developing a temporary targeted therapeutic intervention of HAS3 expression or function in the microcirculation may emerge as a desirable strategy toward tempering colitis in patients undergoing flares of IBD. Sean P. Kessler, Dana R. Obery, and Carol de la Motte Copyright © 2015 Sean P. Kessler et al. All rights reserved. Carboxymethyl Hyaluronan-Stabilized Nanoparticles for Anticancer Drug Delivery Thu, 10 Sep 2015 11:57:28 +0000 Carboxymethyl hyaluronic acid (CMHA) is a semisynthetic derivative of HA that is recognized by HA binding proteins but contains an additional carboxylic acid on some of the 6-hydroxyl groups of the N-acetyl glucosamine sugar units. These studies tested the ability of CMHA to stabilize the formation of calcium phosphate nanoparticles and evaluated their potential to target therapy resistant, CD44+/CD24−/low human breast cancer cells (BT-474EMT). CMHA stabilized particles (nCaPCMHA) were loaded with the chemotherapy drug cis-diamminedichloroplatinum(II) (CDDP) to form nCaPCMHACDDP. nCaPCMHACDDP was determined to be poorly crystalline hydroxyapatite, 200 nm in diameter with a −43 mV zeta potential. nCaPCMHACDDP exhibited a two-day burst release of CDDP that tapered resulting in 86% release by 7 days. Surface plasmon resonance showed that nCaPCMHACDDP binds to CD44, but less effectively than CMHA or hyaluronan. nCaP was taken up by CD44+/CD24− BT-474EMT breast cancer cells within 18 hours. nCaPCMHACDDP was as cytotoxic as free CDDP against the BT-474EMT cells. Subcutaneous BT-474EMT tumors were more reproducibly inhibited by a near tumor dose of 2.8 mg/kg CDDP than a 7 mg/kg dose nCaPCMHACDDP. This was likely due to a lack of distribution of nCaPCMHACDDP throughout the dense tumor tissue that limited drug diffusion. Jessica L. Woodman, Min Sung Suh, Jianxing Zhang, Yuvabharath Kondaveeti, Diane J. Burgess, Bruce A. White, Glenn D. Prestwich, and Liisa T. Kuhn Copyright © 2015 Jessica L. Woodman et al. All rights reserved. Utilization of Glycosaminoglycans/Proteoglycans as Carriers for Targeted Therapy Delivery Thu, 10 Sep 2015 11:49:18 +0000 The outcome of patients with cancer has improved significantly in the past decade with the incorporation of drugs targeting cell surface adhesive receptors, receptor tyrosine kinases, and modulation of several molecules of extracellular matrices (ECMs), the complex composite of collagens, glycoproteins, proteoglycans, and glycosaminoglycans that dictates tissue architecture. Cancer tissue invasive processes progress by various oncogenic strategies, including interfering with ECM molecules and their interactions with invasive cells. In this review, we describe how the ECM components, proteoglycans and glycosaminoglycans, influence tumor cell signaling. In particular this review describes how the glycosaminoglycan hyaluronan (HA) and its major receptor CD44 impact invasive behavior of tumor cells, and provides useful insight when designing new therapeutic strategies in the treatment of cancer. Suniti Misra, Vincent C. Hascall, Ilia Atanelishvili, Ricardo Moreno Rodriguez, Roger R. Markwald, and Shibnath Ghatak Copyright © 2015 Suniti Misra et al. All rights reserved. Selective Activation of Cancer Stem Cells by Size-Specific Hyaluronan in Head and Neck Cancer Thu, 10 Sep 2015 11:27:27 +0000 We determined that human head and neck cancer cells (HSC-3 cell line) contain a subpopulation displaying cancer stem cell (CSC) properties and are very tumorigenic. Specifically, we investigated whether different sizes of hyaluronan (HA) (e.g., 5 kDa, 20 kDa, 200 kDa, or 700 kDa-HA-sizes) play a role in regulating these CSCs. First, we observed that 200 kDa-HA (but not other sizes of HA) preferentially induces certain stem cell marker expression resulting in self-renewal and clonal formation of these cells. Further analyses indicate that 200 kDa-HA selectively stimulates the expression of a panel of microRNAs (most noticeably miR-10b) in these CSCs. Survival protein (cIAP-1) expression was also stimulated by 200 kDa-HA in these CSCs leading to cisplatin resistance. Furthermore, our results indicate that the anti-miR-10 inhibitor not only decreases survival protein expression, but also increases chemosensitivity of the 200 kDa-HA-treated CSCs. These findings strongly support the contention that 200 kDa-HA plays a pivotal role in miR-10 production leading to survival protein upregulation and chemoresistance in CSCs. Together, our findings suggest that selective activation of oncogenic signaling by certain sizes of HA (e.g., 200 kDa-HA) may be instrumental in the formation of CSC functions leading to tumor cell survival and chemoresistance in head and neck cancer progression. Marisa Shiina and Lilly Y. W. Bourguignon Copyright © 2015 Marisa Shiina and Lilly Y. W. Bourguignon. All rights reserved. Roles of Proteoglycans and Glycosaminoglycans in Wound Healing and Fibrosis Thu, 10 Sep 2015 11:25:11 +0000 A wound is a type of injury that damages living tissues. In this review, we will be referring mainly to healing responses in the organs including skin and the lungs. Fibrosis is a process of dysregulated extracellular matrix (ECM) production that leads to a dense and functionally abnormal connective tissue compartment (dermis). In tissues such as the skin, the repair of the dermis after wounding requires not only the fibroblasts that produce the ECM molecules, but also the overlying epithelial layer (keratinocytes), the endothelial cells, and smooth muscle cells of the blood vessel and white blood cells such as neutrophils and macrophages, which together orchestrate the cytokine-mediated signaling and paracrine interactions that are required to regulate the proper extent and timing of the repair process. This review will focus on the importance of extracellular molecules in the microenvironment, primarily the proteoglycans and glycosaminoglycan hyaluronan, and their roles in wound healing. First, we will briefly summarize the physiological, cellular, and biochemical elements of wound healing, including the importance of cytokine cross-talk between cell types. Second, we will discuss the role of proteoglycans and hyaluronan in regulating these processes. Finally, approaches that utilize these concepts as potential therapies for fibrosis are discussed. Shibnath Ghatak, Edward V. Maytin, Judith A. Mack, Vincent C. Hascall, Ilia Atanelishvili, Ricardo Moreno Rodriguez, Roger R. Markwald, and Suniti Misra Copyright © 2015 Shibnath Ghatak et al. All rights reserved.