Guan F, Villegas G, Teichman J, Mundel P, Tufro A
Department of Pediatrics and Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, New York, USA.
Kidney Int. 2006 May;69(9):1564-9. doi: 10.1038/sj.ki.5000313.
Class 3 semaphorins are guidance proteins that play crucial roles during development. Semaphorins 3A (sema 3A) and 3F are expressed by podocytes in vivo throughout ontogeny and their function is unknown. Here we examined the expression of class 3 semaphorins (3A, 3B, 3C, 3D, 3E, and 3F) and their receptors (neuropilins 1 and 2, plexins A1, A2, A3, B2, and D1) in undifferentiated and differentiated mouse podocytes using reverse transcriptase-polymerase chain reaction (RT-PCR). All class 3 semaphorins, neuropilins 1 and 2 are expressed by undifferentiated and differentiated podocytes at similar levels. Differentiated podocytes expressed 2-4-fold higher plexin A1, A2, and A3 mRNA levels than undifferentiated podocytes. To examine semaphorin regulation, we exposed podocytes to recombinant sema 3A. Sema 3A decreased semaphorin 3B, plexin A1, A3, and D1 >/=50% and reduced plexin A2 mRNA to undetectable levels. To identify sema 3A function in podocytes, we examined whether sema 3A regulates slit diaphragm proteins and podocyte survival. Sema 3A induced a dose-response podocin downregulation and decreased its interaction with CD2-associated protein and nephrin, as determined by Western analysis and co-immunoprecipitation. To evaluate sema 3A role in podocyte survival, we quantified podocyte apoptosis using a caspase 3 activity marker. Sema 3A induced a 10-fold increase in podocyte apoptosis and significantly decreased the activity of the Akt survival pathway. Our data indicate that (1) immortalized podocytes in culture have a functional autocrine semaphorin system that is regulated by differentiation and ligand availability; (2) sema 3A signaling regulates the expression and interactions of slit-diaphragm proteins and decreases podocyte survival.
3类信号素是在发育过程中发挥关键作用的导向蛋白。信号素3A（sema 3A）和3F在整个个体发育过程中由足细胞在体内表达，其功能尚不清楚。在这里，我们使用逆转录聚合酶链反应（RT-PCR）检测了未分化和分化的小鼠足细胞中3类信号素（3A、3B、3C、3D、3E和3F）及其受体（神经纤毛蛋白1和2、丛状蛋白A1、A2、A3、B2和D1）的表达。所有3类信号素、神经纤毛蛋白1和2在未分化和分化的足细胞中以相似水平表达。分化的足细胞表达的丛状蛋白A1、A2和A3 mRNA水平比未分化的足细胞高2至4倍。为了检测信号素的调节作用，我们将足细胞暴露于重组sema 3A。sema 3A使信号素3B、丛状蛋白A1、A3和D1降低≥50%，并将丛状蛋白A2 mRNA水平降低至检测不到的水平。为了确定sema 3A在足细胞中的功能，我们检测了sema 3A是否调节裂孔隔膜蛋白和足细胞存活。通过蛋白质免疫印迹分析和免疫共沉淀确定，sema 3A诱导剂量依赖性的足动蛋白下调，并降低其与CD2相关蛋白和nephrin的相互作用。为了评估sema 3A在足细胞存活中的作用，我们使用半胱天冬酶3活性标记物对足细胞凋亡进行了定量。sema 3A诱导足细胞凋亡增加10倍，并显著降低Akt存活途径的活性。我们的数据表明：（1）培养的永生化足细胞具有一个功能性自分泌信号素系统，并受分化和配体可用性的调节；（2）sema 3A信号传导调节裂孔隔膜蛋白的表达和相互作用，并降低足细胞存活。
