A Conditional Knockout Mouse Model Reveals That Calponin-3 Is Dispensable for Early B Cell Development

Calponins form an evolutionary highly conserved family of actin filament-associated proteins expressed in both smooth muscle and non-muscle cells. Whereas calponin-1 and calponin-2 have already been studied to some extent, little is known about the role of calponin-3 under physiological conditions due to the lack of an appropriate animal model. Here, we have used an unbiased screen to identify novel proteins implicated in signal transduction downstream of the precursor B cell receptor (pre-BCR) in B cells. We find that calponin-3 is expressed throughout early B cell development, localizes to the plasma membrane and is phosphorylated in a Syk-dependent manner, suggesting a putative role in pre-BCR signaling. To investigate this in vivo, we generated a floxed calponin-3-GFP knock-in mouse model that enables tracking of cells expressing calponin-3 from its endogenous promoter and allows its tissue-specific deletion. Using the knock-in allele as a reporter, we show that calponin-3 expression is initiated in early B cells and increases with their maturation, peaking in the periphery. Surprisingly, conditional deletion of the Cnn3 revealed no gross defects in B cell development despite this regulated expression pattern and the in vitro evidence, raising the question whether other components may compensate for its loss in lymphocytes. Together, our work identifies calponin-3 as a putative novel mediator downstream of the pre-BCR. Beyond B cells, the mouse model we generated will help to increase our understanding of calponin-3 in muscle and non-muscle cells under physiological conditions.

钙蛋白家族（Calponins）是一类在进化上高度保守的肌动蛋白丝结合蛋白，广泛表达于平滑肌与非肌肉细胞中。尽管钙蛋白-1（calponin-1）与钙蛋白-2（calponin-2）已得到一定程度的研究，但由于缺乏合适的动物模型，目前对生理条件下钙蛋白-3（calponin-3）的功能仍知之甚少。
本研究通过无偏筛选，鉴定出B细胞中前B细胞受体（pre-BCR）下游信号转导通路相关的新型蛋白。本研究发现，钙蛋白-3在B细胞发育的全过程中均有表达，定位于细胞膜，并以脾酪氨酸激酶（Syk）依赖的方式发生磷酸化，提示其可能参与pre-BCR信号转导。
为在体内研究该功能，我们构建了带flox位点的钙蛋白-3-绿色荧光蛋白（GFP）敲入小鼠模型，该模型可通过内源性启动子追踪表达钙蛋白-3的细胞，并可实现其组织特异性敲除。以该敲入等位基因作为报告基因，我们证实钙蛋白-3的表达始于早期B细胞，并随细胞成熟逐渐上调，在外周组织中达到峰值。
令人意外的是，尽管钙蛋白-3的表达具有调控性且体外实验已证实其功能，但条件性敲除Cnn3基因并未导致B细胞发育出现明显异常，这引发了一个疑问：淋巴细胞中是否存在其他蛋白可代偿钙蛋白-3的缺失功能。
综上，本研究证实钙蛋白-3是pre-BCR下游的一种潜在新型调控介质。除B细胞外，我们构建的这款小鼠模型将有助于进一步阐明生理条件下钙蛋白-3在肌肉与非肌肉细胞中的功能。