Glycoconj J (2013) 30:497–510 DOI 10.1007/s10719-012-9450-x Structural remodeling of proteoglycans upon retinoic acid-induced differentiation of NCCIT cells Leyla Gasimli & Hope E. Stansfield & Alison V. Nairn & Haiying Liu & Janet L. Paluh & Bo Yang & Jonathan S. Dordick & Kelley W. Moremen & Robert J. Linhardt Received: 19 July 2012 /Revised: 8 September 2012 /Accepted: 10 September 2012 /Published online: 2 October 2012 # Springer Science+Business Media, LLC 2012 Abstract Pluripotent and multipotent cells become in- retinoic acid-induced changes in the cellular proteogly- creasingly lineage restricted through differentiation. can composition of the well-characterized teratocarcino- Alterations to the cellular proteoglycan composition ma line NCCIT. Our analysis revealed changes in the and structure should accompany these changes to influ- abundance of transcripts for genes encoding core pro- ence cell proliferation, delineation of tissues and acqui- teins, enzymes that are responsible for early and late sition of cell migration capabilities. Retinoic acid plays linkage region biosynthesis, as well as enzymes for an important role in pre-patterning of the early embryo. GAG chain extension and modification. Transcript lev- Retinoic acid can be used in vitro to induce differenti- els for genes encoding core proteins used as backbones ation, causing pluripotent and multipotent cells to be- for polysaccharide synthesis revealed highly significant come increasingly lineage restricted. We examined increases in expression of lumican and decorin, 1,500- Electronic supplementary material The online version of this article (doi:10.1007/s10719-012-9450-x) contains supplementary material, which is available to authorized users. L. Gasimli : J. S. Dordick : R. J. Linhardt H. E. Stansfield Department of Biology, Center for Biotechnology Department of Orthopedics, Center for Muscoloskeletal Research, and Interdisciplinary Studies, Rensselaer Polytechnic Institute, University of Rochester Medical Center, Troy, NY 12180, USA Rochester, NY 14642, USA H. Liu : B. Yang : R. J. Linhardt Department of Chemistry and Chemical Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180, USA A. V. Nairn : K. W. Moremen J. S. Dordick : R. J. Linhardt Complex Carbohydrate Research Center, University of Georgia, Department of Chemical and Biological Engineering, Athens, GA 30602, USA Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180, USA J. S. Dordick : R. J. Linhardt (*) Department of Biomedical Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, J. L. Paluh 110 8th Street, Nanobioscience Constellation, College of Nanoscale Science Troy, NY 12180, USA and Engineering, University at Albany, SUNY, e-mail: [email protected] Albany, NY 12203, USA 498 Glycoconj J (2013) 30:497–510 fold and 2,800-fold, respectively. Similarly, glypican 3, Introduction glypican 5, versican and glypican 6 showed increases be- tween 5 and 70-fold. Significant decreases in biglycan, ser- New strategies in medical therapies, such as induced differ- glycin, glypican 4, aggrecan, neurocan, CD74 and glypican 1 entiation of cancer cells to block metastasis, require the were observed. Disaccharide analysis of the glycans in hepa- ability to redirect cell behavior. In addition, optimizing the rin/heparan sulfate and chondroitin/dermatan sulfate revealed efficacy of cell-based therapies in a multicellular environ- retinoic acid-induced changes restricted to chondroitin/derma- ment will require an understanding of how the cellular tan sulfate glycans. Our study provides the first detailed glycome can respond. In disease states, an inappropriate analysis of changes in the glycosaminoglycan profile of hu- glycome may also provide abnormal cellular capabilities man pluripotent cells upon treatment with the retinoic acid for a given tissue environment by facilitating new signaling morphogen. pathways. Systematic analysis can help to provide a data- base of responses that can predict cellular outcomes. The tissue multicellular environment is complex, requir- Keywords Glycomics . Teratocarcinoma . Pluriotent . ing internal epigenome, transcriptome and proteome Glycosaminoglycans changes that are coordinated to remodel the extracellular glycome. Glycosaminoglycans (GAGs) localize mainly to Abbreviations the external membrane of cells and reside within the extra- Ac Acetyl cellular matrix. They are attached to core proteins to form BCA assay Bicinchoninic acid assay cellular proteoglycans (PGs) that are critical for a variety of BEH Ethylene bridged hybrid cell signaling and migration roles as well as mammalian C5Epi C5Epimerase organ development [1, 2]. GAGs therefore act as molecular cDNA Complementary deoxyribonucleic acid co-receptors in cell signaling for cell–cell interactions and as CHAPS 3-[(3-cholamidopropyl)dimethylammonio]- regulators of cell adhesion, cell growth and differentiation 1-propanesulfonate [2, 3]. By defining temporal changes in GAG profiles on CS/DS Chondroitin sulfate/dermatan sulfate cells during cellular quiescence, proliferation, and lineage ΔUA 4-deoxy-α-L-threo-hex-4-enopryanosyluronic differentiation, we refine our global view of development in acid the multicellular environment. This is particularly critical GAG Glycosaminoglycan for novel therapeutic applications with stem cells or tissue GalN Galactosamine progenitors as well as efforts to optimize the in vitro stem GFAP Glial fibrillary acid protein cell niche for cell, tissue and organ based therapies [2, 3]. GlcA Glucuronic acid Teratocarcinomas are malignant germ cell tumors con- IdoA Iduronic acid sisting of embryonal carcinoma (EC) cells. They are malig- GlcN Glucosamine nant equivalents of normal pluripotent embryonic cells from HRP Horseradish peroxidase the pre-implantation stage of embryos [4, 5]. Single embry- HFIP 1,1,1,3,3,3-hexafluoro-2-propanol onal carcinoma cells from teratocarcinomas develop into HP/HS Heparin/heparan sulfate tumors that contain a mixed population of more than two HXA Hexylamine dozen well-differentiated adult tissues from all three germ KRTAP3-2 Keratin associated protein 3-2 layers, including brain, muscle, bone, teeth, bone marrow, LC/MS Liquid chromatography/mass spectrometry eyes, secretory glands, skin and intestine, as well as placen- NDST N-deacetylase-N-sulfotransferase tal and yolk sac tissue. The ability of in vitro cultured PVDF Polyvinyl difluoride embryonal carcinoma cells to form organized structures that qRT-PCR Quantitative reverse transcription- resemble the developing embryo has promoted their use as a polymerase chain reaction model system for the study of early embryonic development RA Retinoic acid [6]. The widely studied NCCIT cell line, derived from a RPS18 Ribosomal protein S18 mediastinal mixed germ cell tumor, has been shown to RNA Ribonucleic acid differentiate into derivatives of all three embryonic germ S Sulfo layers (ectoderm, mesoderm, and endoderm) and extraem- TrBA Tributylamine bryonic cell lineages [7]. The NCCIT line is responsive to UPLC Ultra-performance liquid chromatography retinoic acid (RA), inducing cellular differentiation accom- WB Western immunoblotting panied by the disappearance of oligosaccharide surface anti- HS2ST 2-O-sulfotransferase gens associated with pluripotency [8]. For these reasons, HS3ST 3-O-sulfotransferase coupled with their ease of manipulation, NCCIT cells are a HS6ST 6-O-sulfotransferase useful model to quantify the concomitant changes to the Glycoconj J (2013) 30:497–510 499 glycan profile upon RA treatment to reveal promotive and/ altered drastically upon differentiation (Fig. 1b). We observed or restrictive changes associated with the action of this flattened cells, including some with branched elongated cyto- morphogen for inducing loss of pluripotency and increased plasmic processes typical of neuronal morphology. Changes lineage restriction. in morphology were accompanied by reduced expression of The biosynthetic pathways and enzymes involved in Oct-3/4 (3-fold) (Fig. 2 and Table 1), which has been shown to GAG biosynthesis are well defined and the enzymes and be a pluripotency marker [3]. The expression of Nestin, a their isoforms have been found to be differentially expressed marker for neural differentiation [16], increased 3-fold sug- in various cell types [9–12]. GAGs are linear, sulfated and gesting proliferation of neural progenitor cells. There were no highly charged heterogeneous polysaccharides consisting of changes in expression of beta-III-tubulin (mature neuronal a repeating disaccharide unit. Polysaccharide length and fine marker) or Olig2 (oligodendrocyte marker) observed, whereas structure, in addition to the placement of protein-binding expression of glial fibrillary acidic protein (GFAP), a type III domains, are critical to the functioning of PGs in cell sig- intermediate filament protein common to cells of the central naling. Four distinct types of GAGs are present in eukary- nervous system (particularly astrocytes) was not detected. otic cells: chondroitin sulfate/dermatan sulfate (CS/DS), Expression of keratin associated protein 3-2 remained un- heparin/heparan sulfate (HP/HS), keratan sulfate, and hyalur- changed. Expression of an example marker of mesodermal onan. Modification of GAG profiles with RA treatment to origin RET9 decreased 6-fold. Expression of MYH6 was low induce the loss of pluripotency and lineage commitment