Suva L J, Towler D A, Harada S, Gaub M P, Rodan G A
Division of Bone and Mineral Metabolism, Beth Israel Hospital, Harvard Medical School, Boston, Massachusetts 02215.
Mol Endocrinol. 1994 Nov;8(11):1507-20. doi: 10.1210/mend.8.11.7877619.
We have previously shown that retinoic acid (RA) induces differentiation in an osteoblastic cell line derived from embryonic rat calvaria and that RA has selective effects on zif268 gene expression in these preosteoblastic cells,distinct from those in more mature osteoblasts. In this study we demonstrate that the RA-dependent transcriptional increase in zif268 gene expression is mediated by the interaction of RA receptors (RARs) with a 17 base pair sequence in the zif268 promoter containing a single half-site motif (GTTCA), identical to each of the direct repeats seen in the RAR beta 2 gene. The sequence appears relatively RA-specific, since the zif268 RA-responsive element is not activated by 1,25-dihydroxyvitamin D3 or thyroid hormone (T3). However, cotransfection of RAR expression vectors and an SV-40 promoter chloramphenicol acetyltransferase (CAT) construct containing the single zif268 RA-responsive motif into CV-1 cells demonstrates that the alpha-, beta-, and gamma-RARs transactivate through this element. Extensive mutagenesis of the zif268 promoter region containing the RA response element (RARE) motif confirms that the transactivation and nuclear protein binding activity of this region requires only the half-site motif. The direct involvement of RAR in this DNA-protein interaction has been demonstrated by competitive gel retardation analysis using consensus RAREs and super-shifting of the DNA-protein complex with mouse alpha- or gamma-RAR monoclonal antibodies. In addition, we found that cell-specific suppression of RA-stimulated zif268 gene expression can be attributed to a 29 base pair nucleotide sequence, located downstream of the RA-responsive region in the zif268 gene. This sequence appears to be bound specifically by nuclear protein(s) from several cell types, including osteoblasts. The presence of this sequence in cis to the zif268 RARE or the consensus beta RARE completely blocks the RA-responsiveness of the zif268 gene in differentiated osteoblasts. These data extend the broad spectrum of RA-responsive sequences necessary for DNA binding and transactivation to include regulation via single RARE half-site motifs and suggest that the lack of RA responsiveness in differentiated osteoblasts may be mediated by cell-specific suppression of gene expression.
我们先前已经表明，视黄酸（RA）可诱导源自胚胎大鼠颅骨的成骨细胞系发生分化，并且RA对这些前成骨细胞中的zif268基因表达具有选择性作用，这与更成熟的成骨细胞中的作用不同。在本研究中，我们证明zif268基因表达中RA依赖性转录增加是由RA受体（RARs）与zif268启动子中一个17个碱基对的序列相互作用介导的，该序列包含一个单一半位点基序（GTTCA），与在RARβ2基因中看到的每个直接重复序列相同。该序列似乎相对具有RA特异性，因为zif268 RA反应元件不会被1,25-二羟基维生素D3或甲状腺激素（T3）激活。然而，将RAR表达载体与含有单个zif268 RA反应基序的SV-40启动子氯霉素乙酰转移酶（CAT）构建体共转染到CV-1细胞中表明，α-、β-和γ-RARs通过该元件反式激活。对含有RA反应元件（RARE）基序的zif268启动子区域进行广泛诱变证实，该区域的反式激活和核蛋白结合活性仅需要半位点基序。使用共有RAREs进行竞争性凝胶阻滞分析以及用小鼠α-或γ-RAR单克隆抗体对DNA-蛋白质复合物进行超迁移，已证明RAR直接参与这种DNA-蛋白质相互作用。此外，我们发现RA刺激的zif268基因表达的细胞特异性抑制可归因于zif268基因中RA反应区域下游的一个29个碱基对的核苷酸序列。该序列似乎被包括成骨细胞在内的几种细胞类型的核蛋白特异性结合。该序列与zif268 RARE或共有βRARE顺式存在会完全阻断分化的成骨细胞中zif268基因的RA反应性。这些数据扩展了DNA结合和反式激活所需的广泛RA反应序列谱，以包括通过单个RARE半位点基序进行的调控，并表明分化的成骨细胞中缺乏RA反应性可能是由基因表达的细胞特异性抑制介导的。
