| Objective Parkinson’s disease (PD) is one of the major neurodegenerative disorders. Although clinical and experimental studies suggest the involvement of protein misfolding, oxidative stress, and mitochondrial dysfunction, the fundamental cause of the disease and its underlying mechanism remain elusive. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN)-induced putative kinase 1 (PINK1), a serine/threonine kinase, plays an important role in the quality control of mitochondria and more than 70 PINK1 mutations have been identified to cause early-onset PD in an autosomal recessive manner. However, the regulation of PINK1 gene expression remains elusive. In the present study, we aimed to understand the transcriptional regulation of the PINK1 gene.Materials and methods 1. Firstly we applied 5’-RACE (rapid amplification of cDNA ends, RACE) assay to specifically amplify 5’end of human PINK1 gene. Switching mechanism at 5’ end of RNA transcript (SMART) method was utilized. The gene specific primer was designed to recognize the+431 to 450bp of human PINK1 gene downstream of the translation start site (ATG). The resulting PCR products were cloned into pcDNA/myc-His A vector for sequencing.2. To find out the functional promoter of human PINK1 gene, we amplified a series of segments, all of them are located at the PINK1 promoter area between-1799 to+26. Dual luciferase assay was performed to reflect the promoter activity and to explore the minimal promoter region.3. We analyzed the potential transcription factors binding with the promoter through using Genomatix and TFSearch softwares. According to the result, the transcription factor NFκB was further studied by EMSA to confirm its binding with PINK1 promoter.4. NFκB p65 expression plasmid pMTF-NFκB p65 was co-transfected with pPINK1-A into HEK293, SH-SY5Y and N2a cell lines. Dual luciferase assay was performed to reveal the regulation of NFκB to PINK 1 promoter activity.5. Plasmid pMTF-NFKB p65 transfection was firstly performed in HEK293 cells following by semi-quantitative rt-PCR and western blot; meanwhile, SH-SY5Y cells were transfected with pMTF-NFKB p65 plasmid or treated with LPS at a concentration of 25ng/ml, western blot was applied to detect PINK1 protein.Results 1. PCR products from 5’SMART-RACE were further cloned into pcDNA4/myc-His vector and then sequenced. The results indicated that the adenine (A) located 91bp upstream of the translational start site (ATG) was the first base after the RNA adapter SMARTer ⅡA Oligonucleotide.2.9 plasmids (pPINK1-Ato pPINK1-Ⅰ) were constructed with different fragments of PINK1 gene promoter regions. Luciferase assay showed pPINK1-A (-1799+26) possessed significant promoter activity (113.47± 10.63 RLU). Furthermore, deletion analysis illustrated that pPINK1-G also maintained high promoter activity (78.44± 4.74 RLU), which would be abolished by further deletion.3. Computer-based transcription factor binding site analysis using Genomatix and TFSearch revealed that the human PINK1 gene promoter contained four NFκB binding elements in the 1.8kb promoter region. EMS A results further confirmed that NFκB binded with the human PINK1 gene promoter and the-1493 to-1474bp of PINK 1 promoter region was the main binding site.4. The plasmid pPINK1-A was co-transfected with NFκB expression plasmid pMTF-NFκB p65 into three different cell lines. The results showed that NFκB overexpression significantly increased PINK1 promoter activity to 3.43±0.55 folds,2.11±.10 folds and 1.63±.01 folds (p< 0.01) in HEK293, SH-SY5Y cells and N2A cells, respectively.5. NFκB expression plasmid was transfected into HEK293 cells and then the cells were subjected to rt-PCR and WB tests. The results showed that NFκB overexpression significantly increased endogenous human PINK1 transcription by 59.51%±15.13%(p<0.01) and expression by 114.53% ±38%(p<0.01). Consistently, the endogenous PINK1 bands (FL-PINK1,Δ1-PINK1 and A2-PINK1) were also up regulated in NFκB overexpressing SH-SY5Y cells by 45.26%±19.58%,41.10±.70% and 48.54±4.50%(p<0.01), respectively. Furthermore, for LPS-treated SH-SY5Y cells, endogenous PINK1 proteins expression level were elevated by 26.77%±2.33%,24.46±2.59% and 83.57±.60%(p<0.001), respectively.Conclusion 1. The transcription start site (TSS) of human PINK1 gene is the adenine (A) which is located at 91bp upstream of the translation start site (ATG).2. The 1825bp fragment from-1799 to+26bp of PINK1 gene contains the functional promoter region of the human PINK1 gene. The region from-78 to+26bp according to TSS, which contains the TSS, possessed the minimal promoter sequence.3. PINK1 gene is tightly regulated and transcription factor NFκB can bind with its promoter.4. Transcription factor NFκB significantly up regulates the human PINK1 gene promoter activity.5. Transcription factor NFκB dramatically increases the human PINK1 gene express in transcription and expression levels. |
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