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

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 The Cytoskeletal Protein RHAMM and ERK1/2 Activity Maintain the Pluripotency of Murine Embryonic Stem Cells Jihong Jiang 0 Pooja Mohan 0 Christopher A. Maxwell 0 0 Department of Pediatrics, Child & Family Research Institute, University of British Columbia , Vancouver, British Columbia , Canada 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. - Hyaluronan (HA) is an extracellular polysaccharide, and HArich hydrogels maintain human embryonic stem (ES) cells in their undifferentiated state [1]. Extracellular receptors for HA are likely responsible for the transmission of intracellular signals that maintain ES cell pluripotency. Indeed, CD44, the major cellular receptor for HA, is critical to the expansion, differentiation, and pluripotency of a wide range of stem cells, including cancer stem cells [2], neural [3], mesenchymal [4] and epidermal stem cells [5]. Moreover, human ES cells also express receptor for hyaluronan mediated motility (RHAMM, encoded by HMMR) [6], a putative and controversial receptor for HA [7]. The expression of RHAMM is dramatically reduced with differentiation in vitro, and the silencing of RHAMM with siRNA disrupts the self-renewal of human ES cells [6]. Thus, the extracellular localization of RHAMM, and its putative engagement of HA, may be a critical regulatory cue that decides the cellular fate of ES cells; such an action for RHAMM would be vital to multiple aspects of stem cell biology, including stem cell niches, tissue engineering, and biomaterials. Originally identified as a peptide in the supernatants from proliferative fibroblasts [8], RHAMM may be passively released to the extracellular space through the cell death that accompanies pathological states, such as cancer and inflammation [9]. However, RHAMM is an intracellular protein that associates with both microtubules and actin microfilaments [10]. RHAMM forms a complex with the dynein molecular motor to localize to the centrosome [11] and enable mitotic spindle assembly [1214], and RHAMM regulates signal transduction at microtubules, including the aurora kinase A (AURKA) [14] and ERK1/2 [15] pathways. Thus, the putative physiological role is not yet clear for RHAMM during the self-renewal of ES cells. Here, we examine the localization and action of RHAMM within mouse ES cells, which contain an insertional mutation in one allele of the Hmmr/RHAMM gene. We do not find that RHAMM is expressed on the cell-surface but rather that RHAMM is a cytoskeletal and mitotic spindle protein in these cells. The hemizygous genomic loss of Hmmr/RHAMM attenuates mouse ES cell pluripotency and we use a smallmolecule screen to discover that the phosphorylation of ERK1/2 and AURKA are elevated and these kinases act as barriers to pluripotency in RHAMM+/- mouse ES cells. Materials and Methods Mouse ES cell Culture RHAMM+/- mouse ES cell-lines (BB0166- MMRRC: 026467UCD; and XP0038- MMRRC: 028514-UCD) with mutational inactivation of one allele for the Hmmr/RHAMM gene and the parental control RHAMM+/+ mouse ES cell-line (E14TG2a) were purchased from the International Gene Trap Consortium through a Mutant Mouse Regional Resource Center (University of California, Davis). Briefly, mouse ES cells were maintained on mitomycin C-treated mouse embryonic fibroblast (MEF) cells in medium consisting of high-glucose Dulbeccos modified Eagles medium (DMEM) supplemented with fetal bovine serum (FBS) 16% (ES-qualified, Invitrogen), L-glutamine 2 mM, non-essential amino acids (NEAA) 0.1 mM, leukemia inhibitory factor (LIF) 1000 U/ml (Chemicon), and 2-mercaptoethanol 143 M. For feeder-free cultures, mouse ES cells were grown on CellBind plates (Corning) in medium consisting of Glasgow minimal essential medium (GMEM) (Sigma) containing FBS 15% (ES-qualified, Invitrogen), L-glutamine 2 mM, sodium pyruvate 1 mM, NEAA 0.1 mM, LIF 1000 U/ml, and 2mercaptoethanol 66 M. Quantitative Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) Total RNA was extracted from cells with a RNeasy mini kit (Qiagen), treated with DNase I (Invitrogen), and converted to cDNA with a high capacity cDNA RT kit (Applied Biosystems). Real-time quantitative RT-PCR were performed as described [16] using the primers listed in Table S1. Immunostaining Cells were grown on coverslips coated with 0.1% gelatin, fixed with 4% paraformaldehyde (PFA) for 15 minutes (min) at room temperature (RT). Cells were permeablized and blocked with 0.3% triton X-100, 10% normal donkey serum, 0.1% BSA in PBS, and incubated with antibody listed in Table S2 at their respective dilutions. For staining the antigen on the cell surface, detergent was omitted from the buffer. Following three washes in PBST, the following secondary antibodies were applied for 1 hour at RT: anti-mouse Alexa-488, anti-mouse Alexa-594, anti-mouse Alexa-647, antirabbit Alexa-488, anti-rabbit Alexa-594, or anti-rabbit Alexa-647 (1:1500, Life Technologies). Coverslips were mounted with Prolong Gold antifade reagent (Life Technologies) and counterstained with DAPI. Confocal microscopy was performed using a FluoView Fv10i confocal laser scanning microscope (Olympus) and images were processed with Olympus Fluoview software. Immunoblotting Cells were lysed in RIPA buffer (25 mM Tris-HCl, pH7.4, 150 mM NaCl, 1% Triton X-100, 0.5% Na deoxycholate, 0.1% SDS) supplemented with Protease inhibitor (Roche) and 2 mM Na 3VO4 and 50 mM NaF. Cell lysates were clarified by centrifugation at 16,000 X g for 10 min at 4oC, and protein concentration was determined by BCA protein assay kit (Thermal Scientific). Cell lysates were mixed with SDS sample buffer, separated by SDS-PAGE, and blotted with the following antibodies: anti-RHAMM (Epitomics), anti-Oct3/4 (StemCell Technologies), anti-NUMB (...truncated)