Molecular mechanism of L-proline induced EPL-cell formation.

During early embryogenesis pluripotent cells of the inner cell mass (ICM) give rise to a second pluripotent cell population known as the primitive ectoderm an obligate developmental intermediate and the substrate for gastrulation. The ICM and primitive ectoderm are distinguished on the basis of morphology, gene expression and differentiation potential. However, the signals and mechanisms involved in the transition form ICM to primitive ectoderm are not understood. Culture of ES cells in the presence of a conditioned medium MEDII leads to a transition of ES cells to a population of pluripotent primitive ectoderm-like (EPL) cells that are the in vitro equivalent of the primitive ectoderm. In terms of EPL cell formation the bioactive component of MEDII was identified as L-proline. In this thesis the molecular mechanism by which L-proline induces EPL-cell formation was elucidated. As well as L-proline, short L-proline containing peptides were also shown to induce EPL-cell formation but different peptides displayed different abilities to induce the transition with some inducing the complete transition and others inducing morphology changes only. The mechanism of L-proline induced EPL-cell formation was shown to be independent of NK receptors. The mechanism of L-proline induced EPL-cell formation, as deduced from the results presented in this thesis, was suggested to involve the internalisation of L-proline via the SAT2 amino acid transporter into ES cells as competitive inhibitors of SAT2 prevented EPL-cell formation. MAPK signalling via the action of MEK1 was implicated in L-proline induced EPL-cell formation as inhibitors of MEK1 prevented EPL-cell morphology, gene expression and differentiation potential in the presence of Lproline. PI3K signalling was implicated in L-proline-induced EPL-cell morphology since PI3K inhibitor L Y294002 maintained domed colonies in the presence of L-proline but failed to maintain an ES-cell gene expression profile and differentiation potential. Both MAPK and PI3K signalling were suggested to lie down-stream of L-proline action since treatment of ES cells with L-proline induced the activation of ERK1/2 and Akt down-stream effectors of MAPK and PI3K signalling respectively. A gene potentially involved in the Pl3K-rnediated rnorphology change was Lefty2. Therefore, the mechanism of L-proline induced EPL-cell formation appears to involve internalisation of L-proline and at least two signalling pathways down-stream of L-proline, which regulate different components of the transition.Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 200

Fibroblast Growth Factor Receptor 2 Phosphorylation on Serine 779 Couples to 14-3-3 and Regulates Cell Survival and Proliferation▿

The fibroblast growth factors (FGFs) exert their diverse (or pleiotropic) biological responses through the binding and activation of specific cell surface receptors (FGFRs). While FGFRs are known to initiate intracellular signaling through receptor tyrosine phosphorylation, the precise mechanisms by which the FGFRs regulate pleiotropic biological responses remain unclear. We now identify a new mechanism by which FGFR2 is able to regulate intracellular signaling and cellular responses. We show that FGFR2 is phosphorylated on serine 779 (S779) in response to FGF2. S779, which lies adjacent to the phospholipase Cγ binding site at Y766, provides a docking site for the 14-3-3 phosphoserine-binding proteins and is essential for the full activation of the phosphatidylinositol 3-kinase and Ras/mitogen-activated protein kinase pathways. Furthermore, S779 signaling is essential for promoting cell survival and proliferation in both Ba/F3 cells and BALB/c 3T3 fibroblasts. This new mode of FGFR2 phosphoserine signaling via the 14-3-3 proteins may provide an increased repertoire of signaling outputs to allow the regulation of pleiotropic biological responses. In this regard, we have identified conserved putative phosphotyrosine/phosphoserine motifs in the cytoplasmic domains of diverse cell surface receptors, suggesting that they may perform important functional roles beyond the FGFRs

Phosphorylation of serine 779 in fibroblast growth factor receptor 1 and 2 by protein kinase Cϵ regulates ras/mitogen-activated protein kinase signaling and neuronal differentiation

The FGF receptors (FGFRs) control a multitude of cellular processes both during development and in the adult through the initiation of signaling cascades that regulate proliferation, survival, and differentiation. Although FGFR tyrosine phosphorylation and the recruitment of Src homology 2 domain proteins have been widely described, we have previously shown that FGFR is also phosphorylated on Ser779 in response to ligand and binds the 14-3-3 family of phosphoserine/threonine-binding adaptor/scaffold proteins. However, whether this receptor phosphoserine mode of signaling is able to regulate specific signaling pathways and biological responses is unclear. Using PC12 pheochromocytoma cells and primary mouse bone marrow stromal cells as models for growth factor-regulated neuronal differentiation, we show that Ser779 in the cytoplasmic domains of FGFR1 and FGFR2 is required for the sustained activation of Ras and ERK but not for other FGFR phosphotyrosine pathways. The regulation of Ras and ERK signaling by Ser779 was critical not only for neuronal differentiation but also for cell survival under limiting growth factor concentrations. PKCϵ can phosphorylate Ser779 in vitro, whereas overexpression of PKCϵ results in constitutive Ser779 phosphorylation and enhanced PC12 cell differentiation. Furthermore, siRNA knockdown of PKCϵ reduces both growth factor-induced Ser779 phosphorylation and neuronal differentiation. Our findings show that in addition to FGFR tyrosine phosphorylation, the phosphorylation of a conserved serine residue, Ser779, can quantitatively control Ras/MAPK signaling to promote specific cellular responses.Ana Lonic, Jason A. Powell, Yang Kong, Daniel Thomas, Jessica K. Holien, Nhan Truong, Michael W. Parker, and Mark A. Guthridg

The amino acid transporter SNAT2 mediates L-proline-induced differentiation of ES cells

There is an increasing appreciation that amino acids can act as signaling molecules in the regulation of cellular processes through modulation of intracellular cell signaling pathways. In culture, embryonic stem (ES) cells can be differentiated to a second, pluripotent cell population, early primitive ectoderm-like cells in response to biological activities within the conditioned medium MEDII. The amino acid l-proline has been identified as a component of MEDII required for ES cell differentiation. Here, we define the primary l-proline transporter on ES and early primitive ectoderm-like cells as sodium-coupled neutral amino acid transporter 2 (SNAT2). SNAT2 uptake of l-proline can be inhibited by the addition of millimolar concentrations of other substrates. The addition of excess amino acids was used to regulate the uptake of l-proline by ES cells, and the effect on differentiation was analyzed. The ability of SNAT2 substrates, but not other amino acids, to prevent changes in morphology, gene expression, and differentiation kinetics suggested that l-proline uptake through SNAT2 was required for ES cell differentiation. These data reveal an unexpected role for amino acid uptake and the amino acid transporter SNAT2 in regulation of pluripotent cells in culture and provides a number of specific, inexpensive, and nontoxic culture additives with the potential to improve the quality of ES cell culture.Boon Siang Nicholas Tan, Ana Lonic, Michael B. Morris, Peter D. Rathjen and Joy Rathje

PTPN14 phosphatase and YAP promote TGFβ signalling in rheumatoid synoviocytes

ObjectiveWe aimed to understand the role of the tyrosine phosphatase PTPN14-which in cancer cells modulates the Hippo pathway by retaining YAP in the cytosol-in fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA).MethodsGene/protein expression levels were measured by quantitative PCR and/or Western blotting. Gene knockdown in RA FLS was achieved using antisense oligonucleotides. The interaction between PTPN14 and YAP was assessed by immunoprecipitation. The cellular localisation of YAP and SMAD3 was examined via immunofluorescence. SMAD reporter studies were carried out in HEK293T cells. The RA FLS/cartilage coimplantation and passive K/BxN models were used to examine the role of YAP in arthritis.ResultsRA FLS displayed overexpression of PTPN14 when compared with FLS from patients with osteoarthritis (OA). PTPN14 knockdown in RA FLS impaired TGFβ-dependent expression of MMP13 and potentiation of TNF signalling. In RA FLS, PTPN14 formed a complex with YAP. Expression of PTPN14 or nuclear YAP-but not of a non-YAP-interacting PTPN14 mutant-enhanced SMAD reporter activity. YAP promoted TGFβ-dependent SMAD3 nuclear localisation in RA FLS. Differences in epigenetic marks within Hippo pathway genes, including YAP, were found between RA FLS and OA FLS. Inhibition of YAP reduced RA FLS pathogenic behaviour and ameliorated arthritis severity.ConclusionIn RA FLS, PTPN14 and YAP promote nuclear localisation of SMAD3. YAP enhances a range of RA FLS pathogenic behaviours which, together with epigenetic evidence, points to the Hippo pathway as an important regulator of RA FLS behaviour

L-proline induces differentiation of ES cells: A novel role for an amino acid in the regulation of pluripotent cells in culture

The development of cell therapeutics from embryonic stem (ES) cells will require technologies that direct cell differentiation to specific somatic cell lineages in response to defined factors. The initial step in formation of the somatic lineages from ES cells, differentiation to an intermediate, pluripotent primitive ectoderm-like cell, can be achieved in vitro by formation of early primitive ectoderm-like (EPL) cells in response to a biological activity contained within the conditioned medium MEDII. Fractionation of MEDII has identified two activities required for EPL cell formation, an activity with a molecular mass of 100 M L-proline and some L-proline-containing peptides resulted in changes in colony morphology, cell proliferation, gene expression, and differentiation kinetics consistent with differentiation toward a primitive ectoderm-like cell. This activity appeared to be associated with L-proline since other amino acids and analogs of proline did not exhibit an equivalent activity. Activation of the mammalian target of rapamycin (mTOR) signaling pathway was found to be necessary but not sufficient for L-proline activity; addition of other activators of the mTOR signaling pathway failed to alter the ES cell phenotype. This is the first report describing a role for amino acids in the regulation of pluripotency and cell differentiation and identifies a novel role for the imino acid L-proline.Jennifer M. Washington, Joy Rathjen, Fernando Felquer, Ana Lonic, Michael D. Bettess, Nancy Hamra, Ljiljana Semendric, Boon Siang Nicholas Tan, Julie-Anne Lake, Rebecca A. Keough, Michael B. Morris and Peter D. Rathje