1,25-DIHYDROXYVITAMIN-D3 INHIBITS TRANSCRIPTION OF TYPE-I COLLAGEN GENES IN THE RAT OSTEO-SARCOMA CELL-LINE ROS 17/2.8
1,25-DIHYDROXYVITAMIN-D3 INHIBITS TRANSCRIPTION OF TYPE-I COLLAGEN GENES IN THE RAT OSTEO-SARCOMA CELL-LINE ROS 17/2.8
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DOI：
10.1210/endo-125-1-327
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发表时间：
1989-07-01
期刊：
影响因子：
4.8
通讯作者：
KREAM, BE
中科院分区：
文献类型：
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作者：
HARRISON, JR;PETERSEN, DN;KREAM, BE
我们以前的研究已经证明，1，25-二羟维生素D3 [1，25-（OH）2D 3）]降低培养的胎鼠颅骨和大鼠骨肉瘤细胞中I型胶原合成和前胶原mRNA的稳态水平。为了确定1，25-（OH）2D 3是否调节I型胶原基因的转录，使用核径流测定法直接测量从ROS 17/2.8细胞分离的核中的转录速率。允许在[32 P]UTP存在下进行转录20 min，此时放射性标记的UTP掺入三氯乙酸可沉淀材料中达到最大。UTP掺入被3 μ g/ml放线菌素-D抑制90%，被1 μ g/ml α-D抑制40%。鹅膏毒肽用1，25-（OH）_2D_3处理ROS 17/2.8细胞，可抑制前胶原基因的转录，并呈浓度和时间依赖性。10 nM 1，25-（OH）2D 3使前胶原转录降低了约50%的对照率，并且这种抑制在1，25-（OH）2D 3处理24 h后达到最大。前胶原转录的抑制对胶原是特异性的，因为总RNA合成和β-胶原蛋白的合成都是通过抑制前胶原的转录来实现的。肌动蛋白转录不受1，25-（OH）2D 3的抑制。1，25-（OH）2D 3降低前胶原转录的幅度与其抑制稳态前胶原mRNA水平相当，表明转录是1，25-（OH）2D 3调节骨细胞中胶原基因表达的主要机制。
Our previous studies have demonstrated that 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3)] reduces type I collagen synthesis and steady state levels of procollagen mRNA in cultured fetal rat calvaria and rat osteosarcoma cells. To determine whether 1,25-(OH)2D3 regulates transcription of type I collagen genes, transcription rates were measured directly in nuclei isolated from ROS 17/2.8 cells using a nuclear run-off assay. Transcription was allowed to proceed in the presence of [32P]UTP for 20 min, at which time incorporation of radiolabeled UTP into trichloroacetic acid-precipitable material was maximal. UTP incorporation was inhibited 90% by 3 .mu.g/ml actinomycin-D and 40% by 1 .mu.g/ml .alpha.-amanitin. Treatment of ROS 17/2.8 cells with 1,25-(OH)2D3 inhibited procollagen gene transcription in a concentration and time dependent manner. Procollagen transcription was reduced by approximately 50% of the control rate by 10 nM 1,25-(OH)2D3, and this inhibition was maximal after 24 h of 1,25-(OH)2D3 treatment. The inhibition of procollagen transcription was specific for collagen, since total RNA synthesis and .beta.-actin transcription were not inhibited by 1,25-(OH)2D3. The magnitude of the decrease of procollagen transcription by 1,25-(OH)2D3 was comparable to its inhibition of steady state procollagen mRNA levels, suggesting that transcription is the predominant mechanism by which 1,25-(OH)2D3 regulates collagen gene expression in bone cells.