The higher-order brain dysfunction in TRH knockout mice

据报道，TRH在大脑中具有多种神经生理作用。为了深入了解这些效应的分子机制，特别是在小脑中，我们试图使用TRH基因敲除小鼠和消减cDNA分析来克隆受TRH调控的cDNAs。通过对野生型和TRH；-/-&gt；小脑进行消减杂交分析，获得了100多个克隆。其中4个克隆是相同的cdc2相关蛋白激酶(PFTAIRE Protein Kinase 1，PFTK1)，此前报道该基因只在脑和睾丸中表达。与野生型相比，添加甲状腺激素的正常甲状腺TRH^-/-&GT；小脑中PFTK1mRNA水平显著降低。在人髓母细胞瘤HTB-185细胞中，TRH以时间和剂量依赖的方式诱导表达TRH受体亚型1mRNA的PFTK1mRNA。此外，8-BR-cGMP处理可显著提高PFTK1mRNA的表达水平，cGMP产生的特异性抑制剂ODQ可完全阻断TRH诱导的PFTK1mRNA表达。此外，一氧化氮合酶特异性抑制剂L-NAME可显著抑制TRH诱导的PFTK1mRNA的表达，而MEK抑制剂PD98059和钙通道阻断剂尼莫地平则不能。这些发现首次证明了神经肽和细胞周期相关肽之间的一条新的通路，PFTK1可能是小脑中通过NO-cGMP途径发挥TRH作用的关键调节因子。

TRH has been reported to possess several neurophysiological actions in the brain. To gain insights into the molecular mechanisms underlying these effects, particularly in the cerebellum, we attempted to clone a cDNA that was regulated by TRH using TRH knockout mice and subtractive cDNA analysis. Over 100 clones obtained by subtractive hybridization analysis between the wild-type and TRH^<-/-> cerebellum were analyzed. Four clones among them were identical and cdc2-related kinase (PFTAIRE protein kinase 1 (PFTK1)) cDNA, which was previously reported to be expressed only in the brain and testis. PFTK1 mRNA levels in the euthyroid TRH^<-/-> cerebellum supplemented with thyroid hormone were significantly decreased compared with those in the wild-type. Induction of PFTK1 mRNA by TRH was also observed in a time- and dose-dependent manner in human medulloblastoma-derived HTB-185 cells that expressed TRH receptor subtype 1 mRNA. In addition, treatment of 8-Br-cGMP significantly increased PFTK1 mRNA levels, and a specific inhibitor of cGMP production, ODQ, completely blocked TRH-induced expression of PFTK1 mRNA. Furthermore, induction of PFTK1 mRNA by TRH was significantly inhibited by a NOS specific inhibitor, L-NAME, but not by a MEK inhibitor, PD98059 or a calcium channel inhibitor, nimodipine. These findings demonstrated, for the first time, a novel pathway between a neuropeptide and a cell cycle related peptide in the brain, and PFTK1 may be a key regulator for TRH action in the cerebellum through the NO-cGMP pathway.