DHRS3, a regulator of retinoic acid metabolism, plays an essential role in osteogenesis

Abstract

Vitamin A is an essential nutrient during both embryonic development and postnatal life. All-trans-retinoic acid (ATRA), an active metabolite of vitamin A mediates a variety of signaling pathways through regulating the expression levels of more than 500 genes by activating its nuclear receptor, retinoic acid receptor (RAR), and its heterodimer retinoid X receptor (RXR) at retinoid acid response elements (RAREs). Any alterations in vitamin A metabolism or signaling pathways can contribute to harmful effects such as congenital disorders, proliferative defects, and toxicity. Therefore, ATRA levels need to be precisely regulated by a complex network of enzymes, binding proteins, and transporters. The primary aims of this study were to uncover the mechanism underlying the regulation of dehydrogenase/reductase 3 (DHRS3), a critical enzyme in ATRA-signaling, and to determine if the alteration in the activity of this enzyme impacts ATRA levels or signaling pathways during the differentiation of osteoprogenitor cells. We employed several molecular and immunoprecipitation techniques to identify responsive regulatory elements upstream of the Dhrs3 gene that bind RARs. Additionally, we developed different cell models to impair ATRA metabolism through both genetic and biochemical methods. Lastly, we exposed cells to mechanical tension using various means to explore the effect of microenvironmental factors on the regulation of ATRA signaling. We disrupted Dhrs3 activity in mouse MC3T3-E1 calvarial preosteoblasts using CRISPR-Cas9 and induced endogenous excess ATRA formation to mimic the human disorder caused by Dhrs3 mutations. Our findings demonstrated that RAR directly induces Dhrs3 expression through a RARE-mediated mechanism that promotes the activation of the transcription complexes. Moreover, our results revealed the detrimental effects of disturbing ATRA-signaling through the administration of WIN 18446, exogenous RA, or disruption of Dhrs3, which all caused significant changes in the process of osteogenic differentiation of preosteoblasts. Additionally, our results provided new insights about the synergistic function of Yes-associated protein (YAP) and RAR in regulating the transcription of RAR target genes.