Role of Lipoprotein Assembly in Maternal-Fetal Transport of Beta-Carotene

胚胎维生素A缺乏或过量与人类和动物模型中的先天性缺陷有关，这是由于胚胎发育期间维生素A的活性形式视黄酸（RA）的转录作用缺陷。β-胡萝卜素（BC）是最丰富的膳食维生素A前体。在胚胎中，BCO 1从母体循环中裂解完整的BC，产生视黄醇，然后氧化为RA。BC被BCO 2不对称裂解，产生β-apo-10 '-胡萝卜素醛（apo 10AL），它可以作为类维生素A（维生素A及其衍生物）的前体，但也可以拮抗RA作用。我们的数据表明，胎盘内合成的脂蛋白介导BC向胎儿的转移。我们推测，apo 10AL，由BC的BCO 2在胎盘中产生，通过HNF 4 α和/或COUP-TF上调微粒体甘油三酯转移蛋白（Mttp; MTP），通过拮抗RA的作用。我们提出了三个具体的目标，以了解在正常母体维生素A状态下BC胎盘-胎儿转移的分子机制。目标1。探讨apo 10AL和RA对胎盘脂蛋白生物合成的调节作用。我们将评估WT、Bco 1-/-和Bco 2-/-小鼠的胎盘类胡萝卜素和类维生素A水平和代谢以及脂蛋白生物合成。目标1A：明确母体单次给予BC后胎盘中发生的类胡萝卜素代谢和脂蛋白产生相关的分子事件。将在BC给药后的不同时间点处死母鼠（在13.5 dpc，通过IP注射）。目标1B：确定妊娠期不同时间单次母体BC给药对类胡萝卜素代谢和脂蛋白产生相关分子事件的影响。在妊娠期间的不同时间对母鼠进行BC给药，并在4和24 h后处死。目标2.确定胎盘MTP缺乏可消除母体给予胚胎BC后的蓄积。将使用Mttp胎盘特异性失活的小鼠模型。在13.5 dpc给予BC后4和24 h处死母鼠，并按照目的1分析胎盘。目标3。探讨apo 10AL和RA对人胎盘脂蛋白生物合成的调节作用。在这个目标中，我们将把我们的研究扩展到人类模型。目的3A：研究apo 10AL对人胎盘组织和Bewo细胞MTP功能的调节作用。我们将：（i）确定BC、apo 10 AL和RA对类胡萝卜素和类维生素A代谢以及脂蛋白生物合成的时间过程和剂量依赖性影响;（ii）通过SiRNA或MTP抑制剂灭活MTP，（iii）通过SiRNA灭活Bco 1和Bco 2;（iv）研究apo 10 AL和RA对MTP表达的竞争作用。目的3B：鉴定Mttp启动子中的顺式元件和apo 10AL介导的Mttp转录增加所需的转录因子。启动子报告基因测定、转录因子敲低和ChIP测定将确定胎盘Mttp表达如何通过HNF 4a和/或COUP-TF受apo 10AL调节。

Embryonic vitamin A deficiency or excess have been linked to congenital defects in humans and animal models, owing to the defective transcriptional action of retinoic acid (RA), the active form of vitamin A, during embryogenesis. β-carotene (BC) is the most abundant dietary vitamin A precursor. In the embryo, BCO1 cleaves intact BC, from the maternal circulation, to generate retinaldehyde, which is then oxidized to RA. Asymmetric cleavage of BC by BCO2, generates β-apo-10'-carotenal (apo10AL) which can serve as a precursor of retinoids (vitamin A and its derivatives), but also antagonize RA action. Our data suggest that lipoproteins synthesized within placenta mediate the transfer of BC towards the fetus. We hypothesize that apo10AL, generated in placenta by BCO2 from BC, upregulates microsomal triglyceride transfer protein (Mttp; MTP) through HNF4α and/or COUP-TFs, by antagonizing the action of RA. We propose three Specific Aims to understand the molecular mechanisms of BC placental-fetal transfer under a normal maternal vitamin A status. Aim 1. To investigate in vivo the regulation of placental lipoprotein biosynthesis by apo10AL and RA. We will assess placental carotenoids and retinoids levels and metabolism, and lipoprotein biosynthesis in WT, Bco1-/- and Bco2-/- mice. Aim 1A: To define the molecular events related to carotenoid metabolism and lipoprotein production that occur in placenta after a single maternal administration of BC. Dams will be sacrificed at various time points post-BC administration (at 13.5 dpc, by IP injection). Aim 1B: To establish the effects of a single maternal BC administration at various times during gestation on the molecular events related to carotenoid metabolism and lipoprotein production. Dams will be administered BC at various times during gestation and sacrificed after 4 and 24 h. Aim 2. To establish that placenta MTP deficiency abrogates accumulation of embryonic BC after its administration to the mother. A mouse model with placental-specific inactivation of Mttp will be used. Dams will be sacrificed 4 and 24 h after BC administration at 13.5 dpc, and placentas analyzed as in Aim 1. Aim 3. To investigate in vitro the regulation of human placental lipoprotein biosynthesis by apo10AL and RA. In this Aim, we will expand our studies to human models. Aim 3A: To study the apo10AL-mediated regulation of MTP function in human placenta explants and Bewo cells. We will: (i) determine the time course and dose-dependent effect of BC, apo10AL and RA on carotenoid and retinoid metabolism and lipoprotein biosynthesis; (ii) inactivate MTP by siRNA or an MTP inhibitor and (iii) Bco1 and Bco2 by siRNA; (iv) study the competing effects of apo10AL and RA on MTP expression. Aim 3B: To identify the cis-elements in the Mttp promoter and the transcription factors required for apo10AL-mediated increase in Mttp transcription. Promoter reporter assays, transcription factors knockdown, and ChIP assays will establish how placental Mttp expression is regulated by apo10AL through HNF4a and/or COUP-TFs.