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The Cytoskeletal Protein RHAMM and ERK1/2 Activity Maintain the Pluripotency of Murine Embryonic Stem Cells

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The Cytoskeletal Protein RHAMM and ERK1/2 Activity Maintain the Pluripotency of Murine Embryonic Stem Cells The Cytoskeletal Protein RHAMM and ERK1/2 Activity Maintain the Pluripotency of Murine Embryonic Stem Cells Jihong Jiang, Pooja Mohan, Christopher A. Maxwell* Department of Pediatrics, Child & Family Research Institute, University of British Columbia, Vancouver, British Columbia, Canada Abstract Receptor for hyaluronan mediated motility (RHAMM, encoded by HMMR) may be a cell-surface receptor for hyaluronan that regulates embryonic stem cell pluripotency and differentiation, however, a precise mechanism for its action is not known. We examined murine embryonic stem cells with and without hemizygous genomic mutation of Hmmr/RHAMM, but we were not able to find RHAMM on the cell-surface. Rather, RHAMM localized to the microtubule cytoskeleton and along mitotic spindles. Genomic loss of Hmmr/RHAMM did not alter cell cycle progression but augmented differentiation and attenuated pluripotency in murine embryonic stem cells. Through a candidate screen of small-molecule kinase inhibitors, we identified ERK1/2 and aurora kinase A as barrier kinases whose inhibition was sufficient to rescue pluripotency in RHAMM+/- murine embryonic stem cells. Thus, RHAMM is not found on the cell-surface of embryonic stem cells, but it is required to maintain pluripotency and its dominant mechanism of action is through the modulation of signal transduction pathways at microtubules. Citation: Jiang J, Mohan P, Maxwell CA (2013) The Cytoskeletal Protein RHAMM and ERK1/2 Activity Maintain the Pluripotency of Murine Embryonic Stem Cells. PLoS ONE 8(9): e73548. doi:10.1371/journal.pone.0073548 Editor: Austin John Cooney, Baylor College of Medicine, United States of America Received April 17, 2013; Accepted July 19, 2013; Published September 3, 2013 Copyright: © 2013 Jiang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: These studies were supported by a Child and Youth Health, New Investigator Award from the SickKids Foundation and the Institute of Human Development Child and Youth Health-CIHR (Grant No. NI11- 005), and by a Pediatric Brain Tumor grant from the Michael Cuccione Foundation for Childhood Cancer Research. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. * E-mail: [email protected] Introduction Originally identified as a peptide in the supernatants from proliferative fibroblasts [8], RHAMM may be passively released Hyaluronan (HA) is an extracellular polysaccharide, and HA- to the extracellular space through the cell death that rich hydrogels maintain human embryonic stem (ES) cells in accompanies pathological states, such as cancer and their undifferentiated state [1]. Extracellular receptors for HA inflammation [9]. However, RHAMM is an intracellular protein are likely responsible for the transmission of intracellular that associates with both microtubules and actin microfilaments signals that maintain ES cell pluripotency. Indeed, CD44, the [10]. RHAMM forms a complex with the dynein molecular motor major cellular receptor for HA, is critical to the expansion, to localize to the centrosome [11] and enable mitotic spindle differentiation, and pluripotency of a wide range of stem cells, assembly [12–14], and RHAMM regulates signal transduction including cancer stem cells [2], neural [3], mesenchymal [4] at microtubules, including the aurora kinase A (AURKA) [14] and epidermal stem cells [5]. Moreover, human ES cells also and ERK1/2 [15] pathways. Thus, the putative physiological express receptor for hyaluronan mediated motility (RHAMM, role is not yet clear for RHAMM during the self-renewal of ES encoded by HMMR) [6], a putative and controversial receptor cells. for HA [7]. The expression of RHAMM is dramatically reduced Here, we examine the localization and action of RHAMM with differentiation in vitro, and the silencing of RHAMM with within mouse ES cells, which contain an insertional mutation in siRNA disrupts the self-renewal of human ES cells [6]. Thus, one allele of the Hmmr/RHAMM gene. We do not find that the extracellular localization of RHAMM, and its putative RHAMM is expressed on the cell-surface but rather that engagement of HA, may be a critical regulatory cue that RHAMM is a cytoskeletal and mitotic spindle protein in these decides the cellular fate of ES cells; such an action for RHAMM cells. The hemizygous genomic loss of Hmmr/RHAMM would be vital to multiple aspects of stem cell biology, including attenuates mouse ES cell pluripotency and we use a small- stem cell niches, tissue engineering, and biomaterials. molecule screen to discover that the phosphorylation of PLOS ONE | www.plosone.org 1 September 2013 | Volume 8 | Issue 9 | e73548 RHAMM and Pluripotency ERK1/2 and AURKA are elevated and these kinases act as 3VO4 and 50 mM NaF. Cell lysates were clarified by barriers to pluripotency in RHAMM+/- mouse ES cells. centrifugation at 16,000 X g for 10 min at 4oC, and protein concentration was determined by BCA protein assay kit Materials and Methods (Thermal Scientific). Cell lysates were mixed with SDS sample buffer, separated by SDS-PAGE, and blotted with the following Mouse ES cell Culture antibodies: anti-RHAMM (Epitomics), anti-Oct3/4 (StemCell RHAMM+/- mouse ES cell-lines (BB0166- MMRRC: 026467- Technologies), anti-NUMB (Abcam), anti-(p) ERK1/2 (Cell UCD; and XP0038- MMRRC: 028514-UCD) with mutational Signaling), anti-ERK (Cell Signaling), anti-AURKA (Abcam), inactivation of one allele for the Hmmr/RHAMM gene and the anti-(p) AURKA (Cell Signaling), and anti-Actin (Sigma). parental control RHAMM+/+ mouse ES cell-line (E14TG2a) were purchased from the International Gene Trap Consortium Fluorescent Activated Cell Sorting Analysis through a Mutant Mouse Regional Resource Center (University Mouse ES cells were harvested by trypsinization and of California, Davis). Briefly, mouse ES cells were maintained washed. For cell surface protein expression, cells were on mitomycin C-treated mouse embryonic fibroblast (MEF) incubated with the primary antibodies (30 min, 4oC), washed cells in medium consisting of high-glucose Dulbecco’s modified twice, and then incubated with PE-conjugated anti-mouse IgM Eagle’s medium (DMEM) supplemented with fetal bovine (BD pharmingen) or anti-rabbit Alexa-647 IgG (30 min, 4oC) serum (FBS) 16% (ES-qualified, Invitrogen), L-glutamine 2 mM, and washed twice. For intracellular protein expression, cells non-essential amino acids (NEAA) 0.1 mM, leukemia inhibitory were fixed and permeabilized with fresh 4% PFA (15 min, RT) factor (LIF) 1000 U/ml (Chemicon), and 2-mercaptoethanol 143 and methanol (10 mins, -20°C), or methanol alone, before µM. For feeder-free cultures, mouse ES cells were grown on immunostaining. The following primary antibodies were used in CellBind plates (Corning) in medium consisting of Glasgow this study: anti-SSEA (StemCell Technologies), anti-N terminal minimal essential medium (GMEM) (Sigma) containing FBS RHAMM (Epitomics), anti-C terminal RHAMM (Epitomics), anti- 15% (ES-qualified, Invitrogen), L-glutamine 2 mM, sodium TPX2 (Novus). For cell cycle analysis, cells were fixed with pyruvate 1 mM, NEAA 0.1 mM, LIF 1000 U/ml, and 2- 70% ethanol at -20°C overnight, and then stained with 60 µg/ml mercaptoethanol 66 µM. propidium iodide (Invitrogen) for 30 min. FACS analysis was performed using a FACSCalibure2 flow cytometer (BD Quantitative Reverse Transcriptase Polymerase Chain biosciences) and the CellQuest software. Reaction (RT-PCR) Total RNA was extracted from cells with a RNeasy mini kit Cell proliferation (Qiagen), treated with DNase I (Invitrogen), and converted to To measure the doubling time for mouse ES cell-lines, 105 cDNA with a high capacity cDNA RT kit (Applied Biosystems). cells were seeded in 24 well CellBind plates. Cell numbers Real-time quantitative RT-PCR were performed as described were counted 24, 48, 72 and 96 hours after plating. Doubling [16] using the primers listed in Table S1. time was calculated by the equation Yend = Ystart × 2(t/T), where T is the doubling time, Ystart is the starting number of Immunostaining ES cells (24 hours after plating) and Yend is the ending Cells were grown on coverslips coated with 0.1% gelatin, number of ES cells after a period of time t. fixed with 4% paraformaldehyde (PFA) for 15 minutes (min) at room temperature (RT). Cells were permeablized and blocked Alkaline Phosphatase (AP) Assays and Embryoid Body with 0.3% triton X-100, 10% normal donkey serum, 0.1% BSA Formation in PBS, and incubated with antibody listed in Table S2 at their Cells were stained with alkaline phosphatase staining kit II respective dilutions. For staining the antigen on the cell (Stemgent) following the manufacture’s instruction. Images surface, detergent was omitted from the buffer. were acquired with a Zeiss Axiovert 200 M inverted Following three washes in PBST, the following secondary microscope. Static suspension culture in knockout DMEM antibodies were applied for 1 hour at RT: anti-mouse (Invitrogen) containing 15% knockout serum replacement Alexa-488, anti-mouse Alexa-594, anti-mouse Alexa-647, anti- (Invitrogen) on ultra low attachment culture dishes was utilized rabbit Alexa-488, anti-rabbit Alexa-594, or anti-rabbit Alexa-647 to form embryoid bodies from Rhamm+/+ and Rhamm+/- ES
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