Ismail Ayesha, Nguyen Cuong V, Ahene Ago, Fleet James C, Uskokovic Milan R, Peleg Sara
Department of Endocrine Neoplasia and Hormonal Disorders, Unit 435, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA.
Mol Endocrinol. 2004 Apr;18(4):874-87. doi: 10.1210/me.2003-0310. Epub 2004 Jan 15.
The vitamin D analog, 1alpha-fluoro-16-ene-20-epi-23-ene-26,27-bishomo-25-hydroxyvitamin D(3) (Ro-26-9228) is tissue selective, with a gene regulation preference for bone over duodenum in vivo. In the human osteoblast-like cells, hFOB, the vitamin D receptor (VDR)-mediated transcriptional potencies of Ro-26-9228 and 1,25-dihydroxyvitamin D(3) (1,25D(3)) were similar, but in the intestinal cells, Caco-2, transcriptional potency of Ro-26-9228 was 10-50 times lower. We hypothesized that transcriptional activation of the VDR by Ro-26-9228 in the two cell types is regulated differently, and compared VDR extracted from hFOB or Caco-2 cells for their abilities to interact with a p160 coactivator [glucocorticoid receptor-interacting protein (GRIP)] and with retinoid X receptor (RXR) by pull-down assays. 1,25D(3) had similar potencies to induce interactions of VDR from the two cell types with these partners of transcription. In contrast, Ro-26-9228 induced interaction of osteoblastic VDR with RXR and GRIP but did not induce these interactions with VDR from Caco-2 cells. Further studies revealed that in hFOB cells the unoccupied VDR was cytoplasmic and proteasome sensitive, and that ligand treatment caused a rapid accumulation of the VDR in the chromatin. Both cytoplasmic and chromatin-associated ligand-bound VDR from hFOB cells had the abilities to interact with GRIP. In contrast, in Caco-2 cells, unoccupied VDR was localized in both the cytoplasm (70%) and the chromatin (30%). In Caco-2 cells, the cytoplasmic VDR was proteasome resistant, and neither 1,25D(3) nor Ro-26-9228 induced its binding to GRIP. Only a small fraction of the chromatin-associated VDR was proteasome sensitive, and this fraction was distinguishable by a faster electrophoretic mobility. 1,25D(3) induced an accumulation of the proteasome-sensitive VDR in the chromatin of Caco-2 cells and binding to GRIP. Ro-26-9228 failed to induce accumulation of the proteasome-sensitive VDR in the chromatin or binding to GRIP, but a coincubation of Caco-2 cells with the analog and a proteasome inhibitor restored these abilities. These results suggest that Ro-26-9228 has poor ability to promote the accumulation of a proteasome-sensitive, transcriptionally active VDR isoform in Caco-2 cells, whereas it does not have this limitation in hFOB cells.
维生素D类似物1α-氟-16-烯-20-表-23-烯-26,27-双高-25-羟基维生素D(3)（Ro-26-9228）具有组织选择性，在体内对骨骼的基因调控偏好高于十二指肠。在人成骨细胞样细胞hFOB中，Ro-26-9228和1,25-二羟基维生素D(3)（1,25D(3)）的维生素D受体（VDR）介导的转录活性相似，但在肠细胞Caco-2中，Ro-26-9228的转录活性低10至50倍。我们推测Ro-26-9228在这两种细胞类型中对VDR的转录激活调控方式不同，并通过下拉实验比较了从hFOB或Caco-2细胞中提取的VDR与p160共激活因子[糖皮质激素受体相互作用蛋白（GRIP）]以及与视黄酸X受体（RXR）相互作用的能力。1,25D(3)诱导这两种细胞类型的VDR与这些转录伴侣相互作用的能力相似。相比之下，Ro-26-9228诱导成骨细胞VDR与RXR和GRIP相互作用，但不诱导其与Caco-2细胞的VDR发生这些相互作用。进一步研究表明，在hFOB细胞中，未占据的VDR位于细胞质中且对蛋白酶体敏感，配体处理导致VDR在染色质中快速积累。来自hFOB细胞的细胞质和与染色质相关的配体结合VDR均具有与GRIP相互作用的能力。相比之下，在Caco-2细胞中，未占据的VDR定位于细胞质（70%）和染色质（30%）中。在Caco-2细胞中，细胞质VDR对蛋白酶体具有抗性，1,25D(3)和Ro-26-9228均未诱导其与GRIP结合。只有一小部分与染色质相关的VDR对蛋白酶体敏感，且这一部分可通过更快的电泳迁移率区分出来。1,25D(3)诱导蛋白酶体敏感的VDR在Caco-2细胞染色质中积累并与GRIP结合。Ro-26-9228未能诱导蛋白酶体敏感的VDR在染色质中积累或与GRIP结合，但将Caco-2细胞与该类似物和蛋白酶体抑制剂共同孵育可恢复这些能力。这些结果表明，Ro-26-9228促进蛋白酶体敏感的、转录活性VDR异构体在Caco-2细胞中积累的能力较差，但在hFOB细胞中不存在这种限制。
