The retinol signaling pathway in mouse pluripotent P19 cells

Abstract

atRA (all-trans-retinoic acid), the active metabolite of retinol (vitamin A), is essential for embryogenesis and maintenance of cellular phenotype in adults. Chemicals that interfere with the metabolism of retinol to atRA, therefore, are a human health concern. During development of a screen for disruptors of this signaling pathway, we investigated whether the mouse pluripotent P19 cell metabolizes retinol to atRA and thus can be used in a cell-based screen for disruptors of the pathway. We found that retinol induced the identical pattern of homeobox gene expression as atRA and its precursor, retinal. Retinol was 160-fold less potent than atRA as an inducer, however. In spite of its lower potency, increased Hoxa1 gene expression was detected 30 min after retinol exposure and increased 40-fold by 2 h. Rdh10 and Aldh1a2/Raldh2, which together convert retinol to atRA in the embryo, were the predominant alcohol and aldehyde dehydrogenases expressed in P19 cells. The cell expressed high mRNA levels of retinol binding proteins, Rbp1 and Rbp4, and the 13,14-dihydroretinol saturase, Retsat. It also expressed all Rar and Rxr isotypes, Crabp1&2, the three Cyp26 genes, and both b-carotene-cleaving genes, Bcmo1 and Bco2. The basal expression levels and retinol responsiveness of 25 pathway-related genes were quantitated by RT-qPCR. A test of the Aldh1a2 inhibitor, citral, showed that the disruption of the pathway was easily detected and quantitated showing that the P19 cell provides an in vitro model system for identifying and exploring the mechanism of action of chemicals that interfere with this critical cellular pathway.

Retinoid Machinery in Distinct Neural Stem Cell Populations with Different Retinoid Responsiveness

“…Interestingly, mRNA for the extracellular ROL carrier (Rbp4) was present in both NE-4C and RGl cells regardless of the stage of their development. Rbp4 expression may contribute to the regulation of vitamin A availability in the proximate environment of neural stem/progenitor cells [27,28] or developing/mature neurons [43,54]. Both retinoid sensitivity and production by differentiating neural stem cells highly depend on the cellular composition of the cultures.…”

A P19 and P19CL6 Cell-Based Complementary Approach to Determine Paracrine Effects in Cardiac Tissue Engineering

“…P19 cells and their derivate P19CL6 cells have already been used to identify the cardiomyogenic potential of single substances and to elucidate signaling pathways. Chen and Reese [2011] made use of P19 cells to study the retinol-signaling pathway and Danalache et al [2010] found a novel cardiomyogenic peptide with its help. Sadek et al [2008] used P19CL6 cells to identify small molecules involved in cardiac fate by screening a chemical library for activators of the signature gene NKX2-5.…”

Transcriptional Regulation of <i>Tal2</i> Gene by All-<i>trans</i> Retinoic Acid (atRA) in P19 Cells

“…[9][10][11] atRA is also known to be capable of inducing embryonic stem cells and embryonal carcinoma cells to differentiate into neural lineages. 12,13) atRA functions as the activating ligand for retinoic acid receptors (RARs), and atRA-RAR complexes regulate the expression of over 500 target genes as a transcription factor. 11,[13][14][15][16] RARs are a member of the nuclear receptor superfamily, 17,18) and consist of three isotypes: α, β, and γ.…”

“…12,13) atRA functions as the activating ligand for retinoic acid receptors (RARs), and atRA-RAR complexes regulate the expression of over 500 target genes as a transcription factor. 11,[13][14][15][16] RARs are a member of the nuclear receptor superfamily, 17,18) and consist of three isotypes: α, β, and γ. In response to atRA signaling, RARs occupy characteristic retinoic acid response elements (RAREs) located in the promoter regions of target genes.…”