Generation and analysis of knock-in mice carrying pseudohypoaldosteronism type II-causing mutations in the cullin 3 gene.
Generation and analysis of knock-in mice carrying pseudohypoaldosteronism type II-causing mutations in the cullin 3 gene.
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DOI：
10.1242/bio.013276
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发表时间：
2015-10-21
期刊：
影响因子：
2.4
通讯作者：
Uchida S
中科院分区：
文献类型：
--
作者：
Araki Y;Rai T;Sohara E;Mori T;Inoue Y;Isobe K;Kikuchi E;Ohta A;Sasaki S;Uchida S
II 型假性醛固酮增多症 (PHAII) 是一种遗传性高血压，由四种不同基因突变引起：无赖氨酸激酶 (WNK) 1 和 4、Kelch 样家族成员 3 (KLHL3) 和 cullin 3 (Cul3)。 Cul3 和 KLHL3 形成 E3 连接酶复合物，可泛素化并降低 WNK4 的表达水平。 PHAII 引起的 WNK4 和 KLHL3 突变会损害 WNK4 泛素化。然而，Cul3突变引起的PHAII的分子发病机制尚不清楚。在培养细胞和人类白细胞中，引起 PHAII 的 Cul3 突变导致外显子 9 的跳跃，产生缺乏 57 个氨基酸的突变 Cul3 蛋白。然而，这种现象是否发生在肾脏中以及是否是体内 PHAII 的发病机制尚不清楚。我们生成了 Cul3 内含子 8 C 末端携带突变 (c.1207−1G>A) 的敲入小鼠，该突变对应于人类 Cul3 基因中引起 PHAII 的突变。杂合的 Cul3G(−1)A/+ 敲入小鼠没有表现出 PHAII 表型，并且外显子 9 的跳跃在它们的肾脏中并不明显。然而，杂合敲入小鼠肾脏中 Cul3 mRNA 表达水平约为野生型小鼠的一半。此外，纯合基因敲入小鼠无法存活。这表明突变等位基因的行为类似于敲除等位基因，并且不会产生缺乏外显子9的Cul3 mRNA。仅Cul3表达的减少不足以在敲入小鼠中产生PHAII。我们的研究结果强调了突变型 Cul3 蛋白的致病作用，并为解释为什么 PHAII 引起的 Cul3 突变会导致肾脏为主的 PHAII 表型提供了见解。摘要：小鼠 Cul3 内含子 8 的敲入突变导致 Cul3 蛋白表达降低。单独降低 Cul3 蛋白表达不会引起 II 型假性醛固酮增多症 (PHAII)。
Pseudohypoaldosteronism type II (PHAII) is a hereditary hypertensive disease caused by mutations in four different genes: with-no-lysine kinases (WNK) 1 and 4, Kelch-like family member 3 (KLHL3), and cullin 3 (Cul3). Cul3 and KLHL3 form an E3 ligase complex that ubiquitinates and reduces the expression level of WNK4. PHAII-causing mutations in WNK4 and KLHL3 impair WNK4 ubiquitination. However, the molecular pathogenesis of PHAII caused by Cul3 mutations is unclear. In cultured cells and human leukocytes, PHAII-causing Cul3 mutations result in the skipping of exon 9, producing mutant Cul3 protein lacking 57 amino acids. However, whether this phenomenon occurs in the kidneys and is responsible for the pathogenesis of PHAII in vivo is unknown. We generated knock-in mice carrying a mutation in the C-terminus of intron 8 of Cul3, c.1207−1G>A, which corresponds to a PHAII-causing mutation in the human Cul3 gene. Heterozygous Cul3G(−1)A/+ knock-in mice did not exhibit PHAII phenotypes, and the skipping of exon 9 was not evident in their kidneys. However, the level of Cul3 mRNA expression in the kidneys of heterozygous knock-in mice was approximately half that of wild-type mice. Furthermore, homozygous knock-in mice were nonviable. It suggested that the mutant allele behaved like a knockout allele and did not produce Cul3 mRNA lacking exon 9. A reduction in Cul3 expression alone was not sufficient to develop PHAII in the knock-in mice. Our findings highlighted the pathogenic role of mutant Cul3 protein and provided insight to explain why PHAII-causing mutations in Cul3 cause kidney-predominant PHAII phenotypes. Summary: A knock-in mutation in intron 8 of Cul3 in mice led to decreased Cul3 protein expression. Decreased Cul3 protein expression alone did not cause pseudohypoaldosteronism type II (PHAII).