In vivo regulation of recombinant cardiac myosin heavy chain gene expression by thyroid hormone.
In vivo regulation of recombinant cardiac myosin heavy chain gene expression by thyroid hormone.
复制标题
DOI：
10.1210/endo.132.3.8440168
复制
发表时间：
1993-03
期刊：
影响因子：
4.8
通讯作者：
K. Ojamaa;Irwin Klein
中科院分区：
文献类型：
--
作者：
K. Ojamaa;Irwin Klein
心肌细胞具有表达外源基因的独特能力，这些基因直接注射到体内的心脏组织中。这项技术使得鉴定负责调节工作心脏中心肌细胞特异性基因的顺式作用 DNA 序列成为可能。在这些研究中，我们将含有α-肌球蛋白重链（α MHC）基因5'侧翼序列的重组质粒引入大鼠心肌，以鉴定足够的启动子/增强子序列，忠实地再现内源基因的活性。 α MHC启动子序列的转录活性通过萤火虫荧光素酶报告基因的活性水平来测量，并报告为相对于共注射的组成型活性病毒启动子构建体（pRSVCAT）的活性，该构建体校正了DNA摄取和转录后事件的变化。我们报道，含有α MHC基因转录起始位点5'侧翼序列-2560至+421碱基对的重组质粒在甲状腺功能减退大鼠心脏中适当失活，其中内源基因的表达也受到抑制。在甲状腺切除大鼠的心脏中，甲状腺激素使该启动子序列的活性增加了 44 倍。相反，尽管该重组质粒在甲状腺功能正常的心肌中具有适当的活性，但其活性不能被甲状腺激素进一步刺激。甲状腺激素对 α MHC 启动子转录活性的调节在甲状腺功能正常和甲状腺功能减退的心脏中是不同的，这表明核调节因子（包括甲状腺激素/类维生素A受体家族）的参与可能根据甲状腺状态而有所不同。
Cardiac myocytes have the unique ability to express exogenous genes that have been injected directly into the heart tissue in vivo. This technique makes it possible to identify cis-acting DNA sequences responsible for the regulation of myocyte-specific genes in a working heart. In these studies we introduced recombinant plasmids containing 5'-flanking sequences of the alpha-myosin heavy chain (alpha MHC) gene into the rat myocardium in order to identify sufficient promoter/enhancer sequences that faithfully reproduced the activity of the endogenous gene. The transcriptional activity of the alpha MHC promoter sequence was measured by the level of activity of the firefly luciferase reporter gene and was reported as the activity relative to a coinjected constitutively active viral promoter construct (pRSVCAT) which corrected for variations in DNA uptake and posttranscriptional events. We report that a recombinant plasmid containing 5'-flanking sequences -2560 to +421 basepairs of the transcriptional start site of the alpha MHC gene was appropriately inactive in the hypothyroid rat heart, in which expression of the endogenous gene was also inhibited. The activity of this promoter sequence was increased 44-fold by thyroid hormone in the hearts of thyroidectomized rats. In contrast, although this recombinant plasmid was appropriately active in the euthyroid myocardium, its activity could not be further stimulated by thyroid hormone. The observation that regulation of the transcriptional activity of the alpha MHC promoter by thyroid hormone was different in euthyroid and hypothyroid hearts suggests that the participation of nuclear regulatory factors, including the thyroid hormone/retinoid family of receptors, may differ according to thyroid status.