Role of h2-calponin in regulating macrophage motility and phagocytosis
Role of h2-calponin in regulating macrophage motility and phagocytosis
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DOI：
10.1074/jbc.m801163200
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发表时间：
2008-09-19
影响因子：
4.8
通讯作者：
Jin, Jian-Ping
中科院分区：
文献类型：
--
作者：
Huang, Qi-Quan;Hossain, M. Moazzem;Jin, Jian-Ping
肌动蛋白细胞骨架在细胞运动中起着重要作用，而细胞运动对于吞噬细胞的功能至关重要。Calponin是一种肌动蛋白相关的调节蛋白。在这里，我们报告了在髓系外周血细胞中发现显著水平的h2亚型的降钙素。为了研究其功能意义，构建了H2-降钙素基因(Cnn2)突变的小鼠。Cnn2-Flox-neo等位基因在两个相互独立的靶向胚胎干细胞克隆中以嵌合体的形式获得了胚系传递。将neo(R)盒插入到Cnn2基因的第二内含子中，导致H2-calponin表达显著下调。用FLP1重组酶去除FRT侧翼的NEO(R)盒挽救了击倒效应。Cre重组酶诱导的loxP侧翼外显子2的缺失消除了H2-calponin蛋白的表达。没有H2-Calponin的小鼠外周血中中性粒细胞和单核细胞数量减少。不含H2的巨噬细胞表现出比H2阳性的巨噬细胞更高的增殖率和更快的迁移速度，这与外周单核细胞和中性粒细胞更快的滞留相一致。在贴壁培养中，不含H2钙蛋白的巨噬细胞的铺展减少，同时肌动蛋白细胞骨架中的原肌球蛋白减少。缺乏H2-Calponin还显著增加了巨噬细胞的吞噬活性，表明了一种新的调节吞噬细胞功能的机制。
The actin cytoskeleton plays a major role in cell motility that is essential for the function of phagocytes. Calponin is an actin-associated regulatory protein. Here we report the finding of significant levels of the h2 isoform of calponin in peripheral blood cells of myeloid lineage. To study the functional significance, h2-calponin gene (Cnn2) interrupted mice were constructed. Germ line transmission of the Cnn2-flox-neo allele was obtained in chimeras from two independent clones of targeted embryonic stem cells. The insertion of the neo(R) cassette into intron 2 of the Cnn2 gene resulted in a significant knockdown of h2-calponin expression. Removing the frt-flanked neo(R) cassette by FLP1 recombinase rescued the knockdown effect. Cre recombinase-induced deletion of the loxP-flanked exon 2 eliminated the expression of h2-calponin protein. H2-calponin-free mice showed reduced numbers of peripheral blood neutrophils and monocytes. H2-calponin-free macrophages demonstrated a higher rate of proliferation and faster migration than that of h2-calponin-positive cells, consistent with a faster diapedesis of peripheral monocytes and neutrophils. H2-calponin-free macrophages showed reduced spreading in adhesion culture together with decreased tropomyosin in the actin cytoskeleton. The lack of h2-calponin also significantly increased macrophage phagocytotic activity, suggesting a novel mechanism to regulate phagocyte functions.