Search

All- trans-retinoic acid (RA), a vitamin A metabolite, is an important signaling molecule required for the proper development of the heart. The epicardium is the main source of RA in the embryonic heart, yet the cardiogenic functions of epicardial-produced RA are not fully understood. Here, we investigated the roles of RA signaling in the embryonic epicardium using in vivo and in vitro models of excess or deficiency of RA. Our results suggested that RA signaling facilitates the cytoskeletal rearrangement required for the epicardial-to-mesenchymal transition of epicardial cells. In vivo treatment with an inhibitor of RA synthesis delayed the migration of epicardial-derived precursor cells (EPDCs) into the myocardium; the opposite was seen in the case of dehydrogenase/reductase superfamily (DHRS)3-deficient embryos, a mouse model of RA excess. Analysis of the behavior of epicardial cells exposed to RA receptor agonists or inhibitors of RA synthesis in vitro revealed that appropriate levels of RA are important in orchestrating the platelet-derived growth factor-induced loss of epithelial character, cytoskeletal remodeling, and migration, necessary for the infiltration of the myocardium by EPDCs. To understand the molecular mechanisms by which RA regulates epicardial cytoskeletal rearrangement, we used a whole transcriptome profiling approach, which in combination with pull-down and inhibition assays, demonstrated that the Ras homolog gene family, member A (RhoA) pathway is required for the morphologic changes induced by RA in epicardial cells. Collectively, these data demonstrate that RA regulates the cytoskeletal rearrangement of epicardial cells via a signaling cascade that involves the RhoA pathway.-Wang, S., Yu, J., Jones, J. W., Pierzchalski, K., Kane, M. A., Trainor, P. A., Xavier-Neto, J., Moise, A. R. Retinoic acid signaling promotes the cytoskeletal rearrangement of embryonic epicardial cells.

Oxidation of retinol via retinaldehyde results in the formation of the essential morphogen all-trans-retinoic acid (ATRA). Previous studies have identified critical roles in the regulation of embryonic ATRA levels for retinol, retinaldehyde, and ATRA-oxidizing enzymes; however, the contribution of retinaldehyde reductases to ATRA metabolism is not completely understood. Herein, we investigate the role of the retinaldehyde reductase Dhrs3 in embryonic retinoid metabolism using a Dhrs3-deficient mouse. Lack of DHRS3 leads to a 40% increase in the levels of ATRA and a 60% and 55% decrease in the levels of retinol and retinyl esters, respectively, in Dhrs3(-/-) embryos compared to wild-type littermates. Furthermore, accumulation of excess ATRA is accompanied by a compensatory 30-50% reduction in the expression of ATRA synthetic genes and a 120% increase in the expression of the ATRA catabolic enzyme Cyp26a1 in Dhrs3(-/-) embryos vs. controls. Excess ATRA also leads to alterations (40-80%) in the expression of several developmentally important ATRA target genes. Consequently, Dhrs3(-/-) embryos die late in gestation and display defects in cardiac outflow tract formation, atrial and ventricular septation, skeletal development, and palatogenesis. These data demonstrate that the reduction of retinaldehyde by DHRS3 is critical for preventing formation of excess ATRA during embryonic development.

Cellular retinol-binding protein, type I (CrbpI), encoded by retinol-binding protein, type 1 (Rbp1), is a chaperone of vitamin A (retinol) that is epigenetically silenced in ~25% of human breast cancers. CrbpI delivers vitamin A to enzymes for metabolism into an active metabolite, all-trans retinoic acid (atRA), where atRA is essential to cell proliferation, apoptosis, differentiation, and migration. Here, we show the effect of CrbpI loss on mammary atRA homeostasis using the Rbp1(-/-) mouse model. Rbp1(-/-) mouse mammary tissue has disrupted retinoid homeostasis that results in 40% depleted endogenous atRA. CrbpI loss and atRA depletion precede defects in atRA biosynthesis enzyme expression. Compensation by CrbpIII as a retinoid chaperone does not functionally replace CrbpI. Mammary subcellular fractions isolated from Rbp1(-/-) mice have altered retinol dehydrogenase/reductase (Rdh) enzyme activity that results in 24-42% less atRA production. Rbp1(-/-) mammary tissue has epithelial hyperplasia, stromal hypercellularity, increased collagen, and increased oxidative stress characteristic of atRA deficiency and early tissue dysfunction that precedes tumor formation. Consistent with the findings from the Rbp1(-/-) mouse, tumorigenic epithelial cells lacking CrbpI expression produce 51% less atRA. Together, these data show that CrbpI loss disrupts atRA homeostasis in mammary tissue, resulting in microenvironmental defects similar to those observed at the early stages of tumorigenesis.