Kindlin-2 deficiency induces fatal intestinal obstruction in mice
Kindlin-2 deficiency induces fatal intestinal obstruction in mice
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Kindlin-2缺陷导致小鼠致命性肠梗阻
DOI：
10.7150/thno.46553
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发表时间：
2020-01-01
期刊：
影响因子：
12.4
通讯作者：
Zhang, Hongquan
中科院分区：
文献类型：
--
作者：
He, Xiaokun;Song, Jiagui;Zhang, Hongquan
原理：平滑肌运动障碍的主要特征是收缩力受损和功能性肠梗阻。其中一些病例是由平滑肌基因ACTA 2、ACTG 2、MYH 11、MYLK和LMOD 1的基因突变引起的。然而，病因是复杂的和多因素的，并且对潜在的病理学知之甚少。整合素相互作用蛋白Kindlin-2广泛表达于横纹肌和平滑肌细胞（SMC）中。然而，Kindlin-2在平滑肌中的功能仍然难以捉摸。研究方法：我们使用不同的cre启动子转基因小鼠，Kindlin-2fl/fl SM 22 α-cre+（cKO小鼠）和Kindlin-2fl/fl; MYH-cre+（iKO小鼠），产生了两种小鼠模型。制备胚胎和成体组织用于苏木精和伊红（H&E）染色、免疫组织化学（IHC）和末端脱氧核苷酸转移酶dUTP缺口末端标记（TUNEL）凋亡测定。用透射电镜观察了小鼠离体主动脉环和肠环平滑肌的超微结构变化，用DMT-620 M型多丝肌电描记系统测定了离体主动脉环和肠环平滑肌收缩力。应用细胞牵引力显微镜（CTFM）观察RNAi去除Kindlin-2后原代SMC的功能变化。结果如下：胚胎平滑肌中Kindlin-2编码基因Fermt 2的缺失导致细胞凋亡，下调SMC的关键组分，损害平滑肌发育，并最终导致E14.5的胚胎死亡。三苯氧胺诱导的Kindlin-2特异性敲除成年小鼠平滑肌表现为血压降低、肠功能减退，最终死于肠梗阻。Kindlin-2缺失还导致iKO小鼠中Myh 11、α-SMA和CNN下调、肌丝缩短、肌原纤维断裂和平滑肌收缩性受损。在机制上，Kindlin-2的缺失通过下调钙结合蛋白S100 A14和STIM 1的表达来减少原代血管平滑肌细胞（PVSMC）中的Ca 2+内流。结论：Kindlin-2对维持平滑肌的正常结构和功能具有重要作用。Kindlin-2的缺失通过诱导细胞凋亡而损害胚胎发育期间的平滑肌形成，并且通过阻断导致肠梗阻的Ca 2+内流而危害成体平滑肌的收缩。成年平滑肌中Kindlin-2缺失的小鼠可能是用于疾病研究、药物治疗和预后的有效的肠梗阻动物模型。
Rationale: Smooth muscle-motility disorders are mainly characterized by impaired contractility and functional intestinal obstruction. Some of these cases are caused by genetic mutations of smooth muscle genes ACTA2, ACTG2, MYH11, MYLK and LMOD1. Still the etiology is complex and multifactorial and the underlying pathology is poorly understood. Integrin interaction protein Kindlin-2 is widely expressed in striated and smooth muscle cells (SMC). However, the function of Kindlin-2 in the smooth muscle remains elusive. Methods: We generated two mouse models using different cre promoter transgenic mice, Kindlin-2fl/fl SM22α-cre+ (cKO mice) and Kindlin-2fl/fl; MYH-cre+ (iKO mice). Embryos and adult tissues were prepared for hematoxylin and eosin (H&E) staining, immunohistochemistry (IHC) and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) apoptosis assay. We investigated ultrastructure changes of mouse smooth muscle using transmission electron microscopy (TEM) and measured smooth muscle contractile force in mounting aortic and intestinal rings using the multiwire myograph system (DMT 620M). In addition, cell traction force microscopy (CTFM) was applied to observe the functional change of primary SMC after Kindlin-2 depletion by RNAi. Results: Depletion of Kindlin-2 encoding gene Fermt2 in embryonic smooth muscles leads to apoptosis, downregulates the key components of SMC, impairs smooth muscle development, and finally causes embryonic death at E14.5. Tamoxifen-induced Kindlin-2-specific knockout in adult mouse smooth muscle showed decreased blood pressure, intestinal hypoperistalsis, and eventually died of intestinal obstruction. Kindlin-2 depletion also leads to downregulated Myh11, α-SMA, and CNN, shortened myofilament, broken myofibrils, and impaired contractility of the smooth muscles in iKO mice. Mechanistically, loss of Kindlin-2 decreases Ca2+ influx in primary vascular smooth muscle cells (PVSMC) by downregulating the expression of calcium-binding protein S100A14 and STIM1. Conclusion: We demonstrated that Kindlin-2 is essential for maintaining the normal structure and function of smooth muscles. Loss of Kindlin-2 impairs smooth muscle formation during embryonic development by inducing apoptosis and jeopardizes the contraction of adult smooth muscle by blocking Ca2+ influx that leads to intestinal obstruction. Mice with Kindlin-2 depletion in adult smooth muscle could be a potent animal model of intestinal obstruction for disease research, drug treatment and prognosis.