Transient modulation of cytoplasmic and nuclear retinoid receptors expression in differentiating human teratocarcinoma NT2 cells.
ABSTRACT Human embryonal carcinoma Ntera2/D1 (NT2) cells treated with retinoic acid (RA) differentiate into several cell types including post-mitotic neurons. In this study we asked if RA-induced differentiation alters the expression of RA and retinol (ROL) binding proteins. The regulation of the intracellular carrier proteins for ROL and RA, cellular retinol binding protein I (CRBP-I), and cellular retinoic acid binding protein I and II (CRABP-I, CRABP-II) were studied along with the nuclear RA receptors RARalpha, RARbeta and RARgamma2. PCR analysis of total mRNA from RA-treated cells showed a biphasic early induction of CRBP-I, CRABP-II, and RARgamma2 genes. The immediate early gene Krox-24, a zinc finger transcription factor which is up-regulated during neuronal differentiation, was also induced, but after 1 week of treatment. The induction of CRBP-I protein synthesis in differentiating NT2 cells was confirmed by western blotting and immunofluorescence experiments. Conversely, the synthetic retinoid N-(4-hydroxyphenyl)retinamide, which induces cell death, but not differentiation in different tumour cell types, did not produce the same modulation on gene expression in NT2 cells. These data suggest that the RA-specific induction of CRBP-I and CRABP-II could be an early event in the process leading to neuronal differentiation of NT2 cells.
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ABSTRACT: Our laboratory is working with the human NTera2/D1 (NT2) cell line which have properties similar to progenitor cells in the CNS. These neural-like precursors cells can differentiate into all three major lineages - neurons, astrocytes, andoligodendrocytes. The pure neuronal population, called the hNT cells, possess characteristics of dopamine (DA) cells. In this dissertation, we performed various experiments to examine the neuronal and dopaminergic development of this cellline. We first cultured our hNT neurons with cells from the developingnigrostriatal (NS) pathway, the ventral mesencephalon and striatum, to determine their influence on survival, neuritic outgrowth, and DA phenotype. The survival ofhNT neurons was substantially greater when they were cultured with embryonicday (E) 18 cells, compared to monocultures or cocultures with either E14 orpostnatal day (P) 1 cells. The neuritic outgrowth of hNT neurons as assessed by the number of primary neurites per cell was increased when cultured with theareas of the brain from E14 and P1. The DA phenotype, as determined by the expression of the rate-limiting enzyme of DA synthesis was not increased in hNTneurons when they were cultured with primary rat cells from the NS pathway.Next we analyzed if the retinoic acid (RA)-treated hNT neurons and the NT2 precursor cells expressed three transcription factors required for development ofthe DA phenotype. We report that NT2 cells endogenously expressed Engrailed-1, Ptx3, and Nurr1 while RA treatment increased Nurr1 but down-regulated Engrailed-1 and Ptx3. Finally, lithium has been shown to stimulate neurogenesisin adult hippocampal precursors as well as influence the Wnt pathway known to be important for the induction of the DA phenotype.
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ABSTRACT: Uterine fibroids are a prevalent gynaecological condition in reproductive-aged women and are the commonest reason for hysterectomy. The cellular composition of clonal fibroids are heterogeneous, with phenotypically dissimilar cells that include smooth muscle cells (SMC), vascular SMC (VSMC) and fibroblasts. The aim of our study was to investigate genes that are commonly differentially expressed between fibroid and myometrial whole tissues in phenotypically different sub-populations of cells isolated from fibroid and myometrium. Genes to be investigated by fluorescence-activated cell sorting, quantitative real-time PCR and immunocytochemistry include transforming growth factor β (TGFB) and retinoic acid (RA) signalling families and steroid hormone receptors. We hypothesised that each cell population isolated from fibroid and myometrium would differ in the expression of fibroid-associated genes. We demonstrated that phenotypically different cellular constituents of uterine fibroids differentially express cellular RA-binding protein 2 (CRABP2), progesterone receptor B (PRB) and TGFB receptor 2 mRNA in fibroid-derived cells of VSMC and SMC phenotype. CRABP2 mRNA was also differentially expressed in fibroblasts and VSMC sub-populations from within clonal fibroid tumours. We conclude that differential regulation of RA, TGFB and PR pathway transcription occurs in fibroid-associated SMC and -fibroblasts and that investigation of paracrine interactions between different cell types within the fibroid microenvironment provides an important new paradigm for understanding the pathophysiology of this common disease.Reproduction 04/2014; 147(5):683-92. DOI:10.1530/REP-13-0580
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ABSTRACT: We analysed the effects of all-trans retinoic acid (ATRA) on proliferation and changes in the global proteome of the nullipotent human embryonal carcinoma cell line 2102Ep and the pluripotent cell line NTERA2 cl.D1 (NT2). Differentially expressed proteins were assessed by 2D-PAGE and mass spectrometry, followed by verification and analysis of protein modifications of proteins of the retinoid pathway. We established a proteome map of the germ cell tumor (GCT) cell line NT2 showing neuronal differentiation under ATRA treatment for 7days. Using bioinformatic analyses, we identified functional groups of altered proteins and potentially involved pathways, of which changes to the organisation of the cytoskeleton and anti-apoptotic effects were the most prominent. Changes observed in the expression of factors involved in the retinoid pathway under ATRA, namely an upregulation of CRBP and CRABP2, were also reflected in GCT tissues of different histologies, providing further insight into factors involved in the differentiation of these pluripotent tumors. Treatment of NT2 germ cell tumor cells with all-trans retinoic acid (ATRA) is a model to investigate differentiation. We analysed differentially expressed proteins by 2D-PAGE and mass spectrometry and provide a proteome map of NT2 cells under 7days of ATRA. By bioinformatic analyses, functional groups of proteins and involved pathways like changes to the cytoskeleton and anti-apoptotic effects were identified. Factors involved in the retinoid pathway, in particular upregulation of CRBP, CRABP1 and CRABP2, also showed differential expression in tumors with different histological subtypes, which provides insight into gene regulation under induced and spontaneous differentiation in germ cell tumors.Journal of proteomics 11/2013; 96. DOI:10.1016/j.jprot.2013.11.010 · 3.93 Impact Factor