The role of lecithin: Retinol acyltransferase (LRAT) in embryonic stem cell spinal motor neuron differentiation

Vitamin A (all-trans-retinol) is an essential vitamin that regulates gene expression and cell differentiation. Retinoic acid (RA) is the principal biologically active retinoid metabolite, and RA regulates gene transcription through retinoic acid and retinoid X receptors. RA signaling serves integral roles in mammalian development, as RA induces neuronal differentiation, and specifies neuronal identity in the neural plate in a spatiotemporal manner. The synthesis and degradation of RA is a tightly regulated process, known as retinoid homeostasis. Lecithin:retinol acyltransferase (LRAT) is an enzyme that contributes to retinoid homeostasis. LRAT catalyzes retinol (ROL) esterification. The generation of retinyl esters (RE) from ROL by LRAT helps maintain retinoid homeostasis by reducing the synthesis of RA from ROL. I am interested in the role of LRAT in retinoid-induced neuronal differentiation, a process that requires RA signaling.;Our laboratory generated LRAT-/- homozygous null mice (LRAT-KO mice), and we used these animals as a negative control to assay Lrat mRNA expression in the spinal cord. We determined that the Lrat gene is expressed in the post-natal mouse spinal cord. This finding suggests that Lrat serves a role in the maintenance of retinoid homeostasis in spinal neurons. We utilized LRAT KO embryonic stem (ES) cells to elucidate the role of the LRAT enzyme in a spinal motor neuron differentiation system.;Embryonic stem cell spinal motor neuron differentiation is induced by the application of exogenous RA in vitro. ROL can also be used to differentiate ES cells into neurons, but the activity of RA synthesizing enzymes retinol dehydrogenase and retinal dehydrogenase are required. ROL esterification by LRAT reduces the generation of RA from ROL, and thus, retinoid-induced ES neuronal differentiation efficiency is affected by the levels of LRAT enzymatic activity. Our hypothesis is that ROL-induced ES neuronal differentiation would be reduced in ES cells that constitutively express Lrat cDNA (LRAT over-expressing (LRAT OE) cells) as compared to LRAT KO cells, which do not esterify ROL. We measured RA-responsive gene transcription after retinoid treatment as a read out of effective RA generation in Wt, LRAT KO, and LRAT OE cells by semi-quantitative PCR. We also used PCR and immunofluorescence staining to assay levels of the spinal motor neuron marker Hb9 to assay differentiation efficiency in the three cell lines. We determined that Hb9+ cell density was increased in LRAT KO cells after ROL treatment, as compared to both Wt and LRAT OE cells. This result supports our hypothesis that LRAT-mediated ROL esterification reduces RA production, which ultimately suppresses RA-responsive gene expression and RA-induced neuronal differentiation.