Molecular cloning of the rat β-carotene 15,15′-monooxygenase gene and its regulation by retinoic acid
Molecular cloning of the rat β-carotene 15,15′-monooxygenase gene and its regulation by retinoic acid
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DOI：
10.1007/s00394-006-0601-3
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发表时间：
2006-05
影响因子：
5
通讯作者：
K. Takitani;Chang-Lin Zhu;Akiko Inoue;H. Tamai
中科院分区：
文献类型：
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作者：
K. Takitani;Chang-Lin Zhu;Akiko Inoue;H. Tamai
背景：β-胡萝卜素具有维生素A原的生物活性，维生素A形成的关键步骤是β-胡萝卜素被一种名为β-胡萝卜素15，15‘-单加氧酶的酶切割成视网膜。近年来，有报道称BCM基因在肠道中的表达受维甲酸(RA)的反馈调节。本研究克隆了编码大鼠BCM基因的全长cDNA，并研究了维生素A缺乏条件下RA对BCM基因在多种组织中表达的调节作用。Northern印迹和逆转录-聚合酶链式反应检测BCM基因的表达。我们还研究了维生素A缺乏大鼠几种组织中BCM基因的表达是否受维甲酸的调节。结果序列分析显示，该克隆有一个1701个碱基的开放阅读框，编码566个氨基酸的蛋白质。预测的多肽与小鼠、人和鸡的BCM分别有94%、81%和66%的同源性。大鼠BCM基因在肠道和肝脏中高表达，而在睾丸、肾脏和肺中表达较弱。免疫印迹显示大鼠骨髓间充质干细胞是一种-kDa蛋白。维生素A缺乏使大鼠小肠Bcm基因表达增加，而全反式维甲酸(ATRA)或9-顺式维甲酸(9-Cisra)可抑制这种上调。维生素A缺乏大鼠肺和睾丸中BCM基因的表达也被ATRA或9-Cisra抑制。然而，肝脏BCM基因的表达仅受ATRA的影响，而肾脏的表达不受维甲酸的影响。由于小肠是β-胡萝卜素转化的主要部位，肠道中bcm基因的表达可能受到更严格的调控。结论RA可能在β-胡萝卜素转化为视网膜的水平下调了多种组织中bcm基因的表达。为了防止过量的视黄醇，动态平衡可能发生在几个组织中β-胡萝卜素转化为视黄醇的水平上。
Backgroundβ-Carotene exhibits biological activity as provitamin A. Key step in vitamin A formation is the cleavage of β-carotene to retinal by an enzyme designated as β-carotene 15,15′-monooxygenase (BCM). Recently, it is reported that expression of BCM gene in the intestine is under feedback regulation by retinoic acid (RA). However, the regulation of BCM gene expression in various other tissues is still unknown.Aim of the studyIn the present study, we identified the full-length cDNA encoding the rat BCM gene and investigated the regulation of its expression in several tissues by RA in the presence of vitamin A deficiency.MethodsWe cloned the full-length cDNA encoding BCM gene from a rat intestinal cDNA library by hybridization screening. The BCM gene expression was examined using Northern blotting and reverse transcription-PCR analysis. We also investigated whether BCM gene expression was regulated by retinoids in several tissues of vitamin A-deficient rats.ResultsSequence analysis of this clone revealed an open reading frame of 1,701 bases encoding a protein of 566 amino acids. The predicted polypeptide showed 94%, 81%, and 66% identity with mouse, human, and chicken BCM, respectively. Rat BCM mRNA was highly expressed in the intestine and liver, while there was weak expression in the testes, kidneys, and lungs. Immunoblotting revealed that rat BCM is a 64-kDa protein. BCM gene expression was increased in the small intestine by vitamin A deficiency compared with that in rats on a control diet, while this upregulation was suppressed by all-transRA (ATRA) or 9-cisRA (9-cisRA). BCM gene expression in the lungs and testes was also suppressed by ATRA or 9-cisRA in rats with vitamin A deficiency. However, hepatic BCM gene expression was only decreased by ATRA and renal expression was not affected by either retinoid. As the small intestine is the major site of β-carotene conversion, intestinal BCM gene expression may be more tightly regulated.ConclusionThese data suggest that BCM gene expression in several tissues may be down-regulated by RA at the level of conversion of β-carotene to retinal. To prevent an excess of retinol, homeostasis may occur at the level of conversion of β-carotene to retinal in several tissues.