Elucidation of novel molecular regulatory mechanisms underlying tyrosine hydroxylase functional expression and development of therapeutics for the CNS neurodegenerative disorders

V-1是一种13 kDa的蛋白，含有4个Ankyrin重复序列，与肌动蛋白帽蛋白CapZ具有特异性的分子相互作用，该蛋白最初被我们鉴定为小鼠出生后脑内的发育调节蛋白。我们最近在体外和体内的研究表明，V-1可以正向控制儿茶酚胺生物合成的限速酶酪氨酸羟基酶(TH)的基因转录，从而促进儿茶酚胺能细胞产生儿茶酚胺。在本研究中，我们试图利用V-1、CAPZ和Pref-1/DLK1的siRNA来阐明V-1调控TH基因表达的分子机制。PC12D是一种内源性表达TH蛋白的大鼠肾上腺嗜铬细胞瘤细胞系，因为在小鼠前脂肪细胞系MC3T3-G2/PA6中，V-1上调了Pref-1/DLK1基因的表达。Western印迹分析显示，下调V-1和CAPZ后，TH蛋白水平下降，同时Pref-1/DLK1蛋白表达下调，而肌动蛋白蛋白水平无明显变化。结果表明，Pref-1/DLK1基因被敲除后，TH蛋白表达下降。此外，报告基因分析表明，通过敲除V-1、CAPZ和Pref-1/DLK1三个基因的siRNAs，TH基因的转录水平降低。这些结果提示，在中枢儿茶酚胺能细胞中，V-1可能通过pref-1/DLK1蛋白与肌动蛋白细胞骨架调节因子CAPZ共同调控TH基因的表达。

V-1 is 13 kDa protein with four ankyrin-repeats for specific molecular interaction with CapZ, an actin capping protein, which was originally identified by us as a developmentally regulated protein in murine postnatal brains. Our recent in vitro and in vivo studies have demonstrated that V-1 can positively control gene transcription of tyrosine hydroxylase (TH), the rate-limiting enzyme for catecholamine biosynthesis, thereby promoting catecholamine production in catecholaminergic cells. In the present study, we tried to elucidate the molecular mechanism underlying control of TH gene expression by V-1 in PC12D cells, a rat adrenal pheochromocytoma cell line endogenously expressing TH protein, using siRNAs for V-1, CapZ and pref-1/DLK1, because the pref-1/DLK1 gene expression is observed to be up-regulated by V-1 in MC3T3-G2/PA6 cells, a mouse preadipocyte cell line. Knockdown of V-1 and CapZ with each siRNA for the both genes resulted in a decrease in TH protein level, accompanied by down-regulation of the expression of pref-1/DLK1 protein, whereas actin protein level showed no changes, as assayed by Western blot analyses. Consistently, it was shown that knockdown of pref-1/DLK1 caused decreased expression of TH protein. Moreover, TH gene transcription is decreased by knockdown of V-1, CapZ and pref-1/DLK1 with each of siRNAs for the three genes, when determined by reporter gene assay. These findings suggest that V-1 may control TH gene expression via pref-1/DLK1 protein in co-operation with CapZ, the actin cytoskeletal regulator, in the CNS catecholaminergic cells.