Suppression of ornithine decarboxylase gene expression by retinoids in cultured human keratinocytes.
Suppression of ornithine decarboxylase gene expression by retinoids in cultured human keratinocytes.
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在培养的人角质形成细胞中类维生素A抑制鸟氨酸脱羧酶基因表达。
DOI：
10.1111/1523-1747.ep12873328
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发表时间：
1990
期刊：
影响因子：
--
通讯作者：
Uitto,J
中科院分区：
文献类型：
--
作者：
Olsen,DR;Hickok,NJ;Uitto,J
在含有 0.15 mM Ca++ 的无血清培养基中维持的人角质形成细胞培养物中，分析了类视黄醇对鸟氨酸脱羧酶 (ODC) 基因表达的调节。将细胞与全反式视黄酸、13-顺式视黄酸或类胡萝卜素 Ro15-0778（10−10 至 10−5M）一起孵育，分离总 RNA，并通过 Northern 杂交和槽印迹与人 ODC cDNA 杂交来分析 ODC 的 mRNA 转录本。细胞处理 24 小时导致 ODC mRNA 水平呈剂量依赖性下降，全反式和 13-顺式视黄酸的 IC50 估计为 ∼1 × 10−8M，而 Ro15-0778 的效果稍差（IC50∼1-5 × 10−7M）。类视黄醇对 ODC mRNA 水平的抑制在孵育约 3 小时时即可检测到，基本上在 12 小时时抑制最大。与 5 × 10−7M 全反式视黄酸孵育 24 小时后，ODC mRNA 水平降低，同时 ODC 酶活性降低。为了确定全反式视黄酸是否直接调节 ODC 基因表达，或者是否需要蛋白质合成，在用蛋白质合成抑制剂处理的培养物中分析 ODC 表达。在放线菌酮或嘌呤霉素存在下，全反式视黄酸不会抑制 ODC mRNA 水平。这些发现表明，ODC 基因表达的抑制并不是全反式视黄酸的直接作用，而是取决于正在进行的蛋白质合成。
Modulation of ornithine decarboxylase (ODC) gene expression by retinoids was analyzed in human keratinocyte cultures maintained in serum-free medium containing 0.15 mM Ca++. Cells were incubated with all-trans-retinoic acid, 13-cis-retinoic acid or arotinoid Ro15-0778 (10−10to 10−5M), total RNA was isolated, and mRNA transcripts for ODC were analyzed by Northern and slot blot hybridizations with a human ODC cDNA. Treatment of cells for 24 h resulted in a dose-dependent decrease in ODC mRNA levels, with an estimated IC50of ∼1 × 10−8M for all-trans- and 13-cis-retinoic acid, while Ro15-0778 was somewhat less effective (IC50∼1-5 × 10−7M). The suppression of ODC mRNA levels by retinoids was detectable at ∼3 h of incubation, with essentially a maximal inhibition at 12 h. Reduced ODC mRNA levels noted after 24 h of incubation with 5 × 10−7M all-trans-retinoic acid were accompanied by a reduction in ODC enzyme activity. To determine if all-trans-retinoic acid was regulating ODC gene expression directly, or if protein synthesis was required, ODC expression was analyzed in cultures treated with protein synthesis inhibitors. In the presence of cycloheximide or puromycin, all-trans-retinoic acid did not suppress ODC mRNA levels. These findings suggest that suppression of ODC gene expression is not a direct effect of all-trans-retinoic acid, but depends on ongoing protein synthesis.