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Tal2 expression is induced by all-trans retinoic acid in P19 cells prior to acquisition of neural fate.

ABSTRACT: TAL2 is a member of the basic helix-loop-helix family and is essential for the normal development of the mouse brain. However, the function of TAL2 during brain development is unclear. P19 cells are pluripotent mouse embryonal carcinoma cells that adopt neural fates upon exposure to all-trans retinoic acid (atRA) and culture in suspension. We found that the expression of Tal2 gene was induced in P19 cells after addition of atRA in suspension culture. Tal2 expression was detected within 3?h after the induction, and had nearly returned to basal levels by 24?h. When GFP-tagged TAL2 (GFP-TAL2) was expressed in P19 cells, we observed GFP-TAL2 in the nucleus. Moreover, we showed that atRA and retinoic acid receptor ? regulated Tal2 expression. These results demonstrate for the first time that atRA induces Tal2 expression in P19 cells, and suggest that TAL2 commits to the acquisition of neural fate in brain development.

SUBMITTER: Kobayashi T

PROVIDER: S-EPMC4017210 | biostudies-literature | 2014 May

REPOSITORIES: biostudies-literature

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Tal2 expression is induced by all-trans retinoic acid in P19 cells prior to acquisition of neural fate.

Kobayashi Takanobu T Komori Rie R Ishida Kiyoshi K Kino Katsuhito K Tanuma Sei-ichi S Miyazawa Hiroshi H

Scientific reports 20140512

TAL2 is a member of the basic helix-loop-helix family and is essential for the normal development of the mouse brain. However, the function of TAL2 during brain development is unclear. P19 cells are pluripotent mouse embryonal carcinoma cells that adopt neural fates upon exposure to all-trans retinoic acid (atRA) and culture in suspension. We found that the expression of Tal2 gene was induced in P19 cells after addition of atRA in suspension culture. Tal2 expression was detected within 3 h after ...[more]

PMID: 24816818

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Project description:The mechanism of action of retinoic acid (RA) is of broad relevance to cell and developmental biology, nutrition, and cancer chemotherapy. RA is known to induce expression of the Burkitt's lymphoma receptor 1 (BLR1) gene which propels RA-induced cell cycle arrest and differentiation of HL-60 human myeloblastic leukemia cells, motivating the present analysis of transcriptional regulation of blr1 expression by RA. The RA-treated HL-60 cells used here expressed all RA receptor (RAR) and retinoid X receptor (RXR) subtypes (as detected by Northern analysis) except RXRgamma. Treatment with RAR- and RXR-selective ligands showed that RARalpha synergized with RXRalpha to transcriptionally activate blr1 expression. A 5'-flanking region capable of supporting RA-induced blr1 activation in HL-60 cells was found to contain a 205-bp sequence in the distal portion that was necessary for transcriptional activation by RA. Within this sequence DNase I footprinting revealed that RA induced binding of a nuclear protein complex to an element containing two GT boxes. Electromobility shift assays (EMSAs) and supershift assays showed that this element bound recombinant RARalpha and RXRalpha. Without RA there was neither complex binding nor transcriptional activation. Both GT boxes were needed for binding the complex, and mutation of either GT box caused the loss of transcriptional activation by RA. The ability of this cis-acting RAR-RXR binding element to activate transcription in response to RA also depended on downstream sequences where an octamer transcription factor 1 (Oct1) site and a nuclear factor of activated T cells (NFATc) site between this element and the transcriptional start, as well as a cyclic AMP response element binding factor (CREB) site between the transcriptional start and first exon of the blr1 gene, were necessary. Each of these sites bound its corresponding transcription factor. A transcription factor-transcription factor binding array analysis of nuclear lysate from RA-treated cells indicated several prominent RARalpha binding partners; among these, Oct1, NFATc3, and CREB2 were identified by competition EMSA and supershift and chromatin immunoprecipitation assays as components of the complex. RA upregulated expression of these three factors. In sum the results of the present study indicate that RA-induced expression of blr1 expression depends on a novel RA response element. This cis-acting element approximately 1 kb upstream of the transcriptional start consists of two GT boxes that bind RAR and RXR in a nuclear protein complex that also contains Oct1, NFATc3, and CREB2 bound to their cognate downstream consensus binding sites.

Project description:PurposeAll-trans retinoic acid (ATRA) plays an important role in ocular development. Previous studies found that retinoic acid could influence the metabolism of scleral remodeling by promoting retinal pigment epithelium (RPE) cells to secrete secondary signaling factors. The purpose of this study was to investigate whether retinoic acid affected secretion of bone morphogenetic protein 2 (BMP-2) and matrix metalloproteinase 2 (MMP-2) and to explore the signaling pathway of retinoic acid in cultured acute retinal pigment epithelial 19 (ARPE-19) cells.MethodsThe effects of ATRA (concentrations from 10-9 to 10-5 mol/l) on the expression of retinoic acid receptors (RARs) in ARPE-19 cells were examined at the mRNA and protein levels using reverse transcription-polymerase chain reaction (RT-PCR) and western blot assay, respectively. The effects of treating ARPE-19 cells with ATRA concentrations ranging from 10-9 to 10-5 mol/l for 24 h and 48 h or with 10-6mol/l ATRA at different times ranging from 6h to 72h were assessed using real-time quantitative PCR (qPCR) and enzyme-linked immunosorbent assay (ELISA). The contribution of RARβ-induced activation of ARPE-19 cells was confirmed using LE135, an antagonist of RARβ.ResultsRARβ mRNA levels significantly increased in the ARPE-19 cells treated with ATRA for 24h and 48h. These increases in RARβ mRNA levels were dose dependent (at concentrations of 10-9 to 10-5 mol/l) with a maximum effect observed at 10-6 mol/l. There were no significant changes in the mRNA levels of RARα and RARγ. Western blot assay revealed that RARβ protein levels were increased significantly in a time-dependent manner in ARPE-19 cells treated with 10-6 mol/l ATRA from 12 h to 72 h, with a marked increase observed at 24 h and 48 h. The upregulation of RARβ and the ATRA-induced secretion in ARPE-19 cells could be inhibited by the RARβ antagonist LE135.ConclusionATRA induced upregulation of RARβ in ARPE-19 cells and stimulated these cells to secrete BMP-2 and MMP-2.

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Tal2 expression is induced by all-trans retinoic acid in P19 cells prior to acquisition of neural fate.

Publications

Tal2 expression is induced by all-trans retinoic acid in P19 cells prior to acquisition of neural fate.

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