Mills K J, Vollberg T M, Nervi C, Grippo J F, Dawson M I, Jetten A M
National Institute of Environmental Health Sciences, NIH, Research Triangle Park, North Carolina 27709, USA.
Cell Growth Differ. 1996 Mar;7(3):327-37.
Retinoic acid (RA) is a potent inducer of differentiation of embryonal carcinoma PCC4.aza1R cells into mesenchymal stem cells. Induction of Hoxa-1, Hoxa-5, cellular retinoic acid-binding protein (CRABP) I and II, and retinoic acid receptor (RAR)-beta expression occurs early in this multistage program of differentiation. RA is also a potent inducer of these genes in the differentiation-defective mutant PCC4(RA)-1; however, RA is much less effective in the mutant cell line PCC4(RA)-2. The up-regulation of several of these genes by RA is, at least in part, due to increased transcription. It is likely that some of these changes are mediated either directly or indirectly by nuclear retinoid receptors. Previously, we characterized the expression of RARs in PCC4.aza1R and (RA)-1 and (RA)-2 cells. In this study, we show that these cells also express retinoid X receptor (RXR)-alpha, RXR-beta, and RXR-gamma and that RA treatment down-regulates the expression of RXR-gamma. No large differences were found in RXR mRNA expression between parental and mutant cell lines except that PCC4(RA)-1 cells expressed an 8-fold higher level of RXR gamma mRNA than the parental cells. To obtain more insight into the retinoid signaling pathways involved in the regulation of this pathway of differentiation, we examined the action of two retinoid receptor-selective agonists and one antagonist. The RAR-selective retinoid SRI-6751-84 is a very effective inducer of transactivation of beta RARE-tk-LUC, but not of RXRE-tk-CAT, in PCC4.aza1R cells and is a very potent inducer of morphological differentiation and Hoxa-1, Hoxa-5, CRABP II, and RAR-beta expression. In contrast, the RXR-selective retinoid SR11,217, which transactivates the RXRE-tk-CAT effectively, but beta RARE-tk-LUC poorly, is unable to induce differentiation and has little effect on the expression of these early genes. The RAR-alpha-selective antagonist Ro 41-5253, which inhibits RARE-dependent transcriptional activation, has by itself no effect on the differentiation of PCC4.aza1R cells. However, this antagonist is able to block the induction of morphological differentiation by the RAR-selective retinoid as well as the expression of Hoxa-1, Hoxa-5, CRABP II, and RAR-beta. Our data suggest that the activation of RAR signaling pathways is important in initiating the cascade of changes in gene expression that result in the differentiation of PCC4.aza1R into mesenchymal stem cells. In addition, we demonstrate that the two mutant cell lines, PCC4(RA)-1 and PCC4(RA)-2, are defective at different stages of the differentiation program.
视黄酸（RA）是诱导胚胎癌PCC4.aza1R细胞分化为间充质干细胞的有效诱导剂。在这个多阶段分化程序的早期，会出现Hoxa - 1、Hoxa - 5、细胞视黄酸结合蛋白（CRABP）I和II以及视黄酸受体（RAR）-β表达的诱导。RA也是分化缺陷型突变体PCC4(RA)-1中这些基因的有效诱导剂；然而，RA在突变细胞系PCC4(RA)-2中的效果要差得多。RA对这些基因中几个基因的上调至少部分是由于转录增加。这些变化中的一些可能是由核类视黄醇受体直接或间接介导的。此前，我们已对PCC4.aza1R以及(RA)-1和(RA)-2细胞中RAR的表达进行了表征。在本研究中，我们表明这些细胞也表达类视黄醇X受体（RXR）-α、RXR-β和RXR-γ，并且RA处理会下调RXR-γ的表达。除了PCC4(RA)-1细胞中RXRγ mRNA的表达水平比亲代细胞高8倍外，在亲代和突变细胞系之间未发现RXR mRNA表达有很大差异。为了更深入了解参与该分化途径调控的类视黄醇信号通路，我们研究了两种类视黄醇受体选择性激动剂和一种拮抗剂的作用。RAR选择性类视黄醇SRI-6751-84是PCC4.aza1R细胞中βRARE-tk-LUC反式激活的非常有效的诱导剂，但不是RXRE-tk-CAT的诱导剂，并且是形态分化以及Hoxa-1、Hoxa-5、CRABP II和RAR-β表达的非常有效的诱导剂。相比之下，RXR选择性类视黄醇SR11,217能有效反式激活RXRE-tk-CAT，但对βRARE-tk-LUC的作用较差，它无法诱导分化，并且对这些早期基因的表达几乎没有影响。RAR-α选择性拮抗剂Ro 41-5253可抑制RARE依赖性转录激活，其本身对PCC赤霉素4.aza1R细胞的分化没有影响。然而，这种拮抗剂能够阻断RAR选择性类视黄醇诱导的形态分化以及Hoxa-1、Hoxa-5、CRABP II和RAR-β的表达。我们的数据表明，RAR信号通路的激活对于启动导致PCC4.aza1R分化为间充质干细胞的基因表达变化级联很重要。此外，我们证明了两种突变细胞系PCC4(RA)-1和PCC4(RA)-2在分化程序的不同阶段存在缺陷。
