Regulation of 130-kDa Smooth Muscle Myosin Light Chain Kinase Expression by an Intronic CArG Element
Regulation of 130-kDa Smooth Muscle Myosin Light Chain Kinase Expression by an Intronic CArG Element
复制标题
DOI：
10.1074/jbc.m113.510362
复制
发表时间：
2013-11-29
影响因子：
4.8
通讯作者：
Herring, B. Paul
中科院分区：
文献类型：
--
作者：
Chen, Meng;Zhang, Wenwu;Herring, B. Paul
背景：MLCK的转录调控机制尚不清楚。结果：从Mylk1基因中删除Carg元件，特异性地降低了130 kDa smMLCK亚型的表达，导致肠收缩和增殖能力下降。结论：130 kDa的SMMLCK亚型具有220 kDa的MLCK所不能弥补的功能。意义：Mylk1小鼠允许检测130 kDa的smMLCK的特定功能。myk1基因编码220 kDa的非肌肉肌球蛋白轻链激酶(MLCK)，130 kDa的平滑肌MLCK(SmMLCK)，以及非催化产物端粒蛋白。总而言之，这些蛋白质在调节平滑肌收缩能力方面起着关键作用。它们的表达变化与许多病理条件有关，因此，了解Mylk1基因转录本的表达调控机制非常重要。在此之前，我们报道了一个高度保守的Carg盒，它结合了血清反应因子，位于Mylk1的第15内含子。因为这个Carg元件靠近驱动130 kDa smMLCK转录的启动子，所以我们研究了它在调节该转录物表达中的作用。结果表明，α-半乳糖苷酶报告基因内含子Carg区域的缺失在体内取消了转基因在小鼠体内的表达。从内源性Mylk1基因中删除Carg区域，特别是在平滑肌细胞中，130 kDa的smMLCK的表达减少了40%，而220 kDa的MLCK或telokin的表达不受影响。这种130 kDa smMLCK表达的减少导致肌球蛋白轻链的磷酸化降低，平滑肌收缩能力减弱，小肠长度减少24%，这与Ki67阳性的平滑肌细胞显著减少有关。总体而言，这些数据表明，myk1基因内含子15中的Carg元件是130 kDa smMLCK最大表达所必需的，而130 kDa smMLCK亚型是调节平滑肌收缩和小肠平滑肌细胞增殖所必需的。
Background: Mechanisms regulating transcription of MLCK are poorly defined. Results: Deleting a CArG element from the mylk1 gene specifically decreased expression of the 130-kDa smMLCK isoform, resulting in decreased intestinal contractility and proliferation. Conclusion: The 130-kDa smMLCK isoform has functions that cannot be compensated for by the 220-kDa MLCK. Significance: Floxed mylk1 mice permit specific functions of the 130-kDa smMLCK to be determined.The mylk1 gene encodes a 220-kDa nonmuscle myosin light chain kinase (MLCK), a 130-kDa smooth muscle MLCK (smMLCK), as well as the non-catalytic product telokin. Together, these proteins play critical roles in regulating smooth muscle contractility. Changes in their expression are associated with many pathological conditions; thus, it is important to understand the mechanisms regulating expression of mylk1 gene transcripts. Previously, we reported a highly conserved CArG box, which binds serum response factor, in intron 15 of mylk1. Because this CArG element is near the promoter that drives transcription of the 130-kDa smMLCK, we examined its role in regulating expression of this transcript. Results show that deletion of the intronic CArG region from a -galactosidase reporter gene abolished transgene expression in mice in vivo. Deletion of the CArG region from the endogenous mylk1 gene, specifically in smooth muscle cells, decreased expression of the 130-kDa smMLCK by 40% without affecting expression of the 220-kDa MLCK or telokin. This reduction in 130-kDa smMLCK expression resulted in decreased phosphorylation of myosin light chains, attenuated smooth muscle contractility, and a 24% decrease in small intestine length that was associated with a significant reduction of Ki67-positive smooth muscle cells. Overall, these data show that the CArG element in intron 15 of the mylk1 gene is necessary for maximal expression of the 130-kDa smMLCK and that the 130-kDa smMLCK isoform is specifically required to regulate smooth muscle contractility and small intestine smooth muscle cell proliferation.