HPRT Deficiency Coordinately Dysregulates Canonical Wnt and Presenilin-1 Signaling: A Neuro-Developmental Regulatory Role for a Housekeeping Gene?
HPRT Deficiency Coordinately Dysregulates Canonical Wnt and Presenilin-1 Signaling: A Neuro-Developmental Regulatory Role for a Housekeeping Gene?
复制标题
DOI：
10.1371/journal.pone.0016572
复制
发表时间：
2011-01-28
期刊：
影响因子：
3.7
通讯作者：
Friedmann, Theodore
中科院分区：
文献类型：
--
作者：
Kang, Tae Hyuk;Guibinga, Ghiabe-Henri;Friedmann, Theodore
我们已经使用了基于微阵列的方法的全球基因表达与定量PCR和蛋白质印迹分析，以确定在人类成纤维细胞和SH-SY 5 Y神经母细胞瘤细胞中的基因失调和异常的细胞过程，通过转导稳定表达针对HPRT的shRNA的逆转录病毒使HPRT缺陷。通过基因本体（GO）和基因集富集分析（GSEA）对微阵列表达数据的分析以及通过GeneSpring GX 10和Panther分类系统的显著途径分析揭示，HPRT缺陷伴随着已知调节神经发生或涉及神经退行性疾病的多种途径中的畸变，包括经典的Wnt/β-连环蛋白和阿尔茨海默病/早老素信号通路。Wnt/β-连环蛋白途径的失调通过蛋白质印迹证明在SH-SY 5 Y细胞向神经元表型的体外分化期间β-连环蛋白的胞质隔离来证实。我们还证明了已知由Wnt信号调节并对多巴胺能神经元的产生和功能至关重要的两个关键转录因子基因;即，Lmx 1a和Engrailed 1在HPRT敲低的SH-SY 5 Y细胞中下调。除了Wnt信号畸变，我们发现早老素-1的表达在HPRT缺陷的SH-SY 5 Y细胞中显示出严重的异常表达，这反映在敲低细胞中早老素-1的23 kDa C-末端片段的显著缺陷。两名LND患者的原代成纤维细胞培养物的Western印迹分析也显示出早老素-1表达失调，包括早老素-1的异常蛋白水解加工。Wnt信号和早老素-1表达失调以及多巴胺能转录因子表达受损的这些证据揭示了HPRT表达所发挥的广泛的多效神经调节缺陷，并为研究LND异常神经发生和神经病理学机制以及恢复这种神经发育缺陷中有效信号的潜在新靶点提出了新的方向。
We have used microarray-based methods of global gene expression together with quantitative PCR and Western blot analysis to identify dysregulation of genes and aberrant cellular processes in human fibroblasts and in SH-SY5Y neuroblastoma cells made HPRT-deficient by transduction with a retrovirus stably expressing an shRNA targeted against HPRT. Analysis of the microarray expression data by Gene ontology (GO) and Gene Set Enrichment Analysis (GSEA) as well as significant pathway analysis by GeneSpring GX10 and Panther Classification System reveal that HPRT deficiency is accompanied by aberrations in a variety of pathways known to regulate neurogenesis or to be implicated in neurodegenerative disease, including the canonical Wnt/beta-catenin and the Alzheimer's disease/presenilin signaling pathways. Dysregulation of the Wnt/beta-catenin pathway is confirmed by Western blot demonstration of cytosolic sequestration of beta-catenin during in vitro differentiation of the SH-SY5Y cells toward the neuronal phenotype. We also demonstrate that two key transcription factor genes known to be regulated by Wnt signaling and to be vital for the generation and function of dopaminergic neurons; i.e., Lmx1a and Engrailed 1, are down-regulated in the HPRT knockdown SH-SY5Y cells. In addition to the Wnt signaling aberration, we found that expression of presenilin-1 shows severely aberrant expression in HPRT-deficient SH-SY5Y cells, reflected by marked deficiency of the 23 kDa C-terminal fragment of presenilin-1 in knockdown cells. Western blot analysis of primary fibroblast cultures from two LND patients also shows dysregulated presenilin-1 expression, including aberrant proteolytic processing of presenilin-1. These demonstrations of dysregulated Wnt signaling and presenilin-1 expression together with impaired expression of dopaminergic transcription factors reveal broad pleitropic neuro-regulatory defects played by HPRT expression and suggest new directions for investigating mechanisms of aberrant neurogenesis and neuropathology in LND and potential new targets for restoration of effective signaling in this neuro-developmental defect.