神經滋養因子BDNF在PC12細胞中與蛋白激酶CK2對SRE所調控之基因轉錄作用的機制探討

Abstract

神經系統裡，神經滋養因子在調控細胞分化與生存作用中扮演非常重要的角色，近年來的研究顯示 BDNF 的神經保護效果是透過細胞外訊息調控激酶 (extracellular signal-regulated kinase, ERK) 及磷脂酰肌醇3-激酶 (phosphatidylinositol-3 kinase, PI3K) 訊息傳遞路徑調控，然而，還有許多其他的細胞信號傳遞路徑可能參與 BDNF 的保護作用機制中。而蛋白激酶 CK2 (casein kinase 2) 是一種普遍存在於細胞且具有高度保留序列的絲胺酸/蘇胺酸蛋白質激酶，在細胞中具有非常重要的地位。近來研究有非常多證據支持 CK2 是細胞凋亡的抑制者。此外，血清反應因子 (serum response factor, SRF) 是一種轉錄因子，會與保留序列 SRE (即 CArG box) 相結合，而此段序列過去曾在早期即時表現基因 (如 c-fos、Egr ) ，或是抗細胞凋亡基因－Mcl-1－上的啟動子被發現， SRF 調控著基因的活化，進而與細胞增生、存活、突觸活性相關聯，然而調節 SRE 調控之基因的作用機制尚未十分明瞭。因此，本論文研究主要探討在 PC12 細胞中， BDNF調節 SRE 調控轉錄作用機轉 CK2 是否參與其中? 由冷光酶活性試驗結果顯示 BDNF 會顯著地促進 SRE 的轉錄活性，並且當 CK2α 過度表現亦會促進 SRE 調控的轉錄活性，而利用小干擾 RNA 抑制內生性 CK2α 生成，則會降低 SRE 的轉錄活性，更進一步證明 CK2α siRNA 會降低 BDNF 促進 SRE 調控的轉錄活性。此外，將 CK2α 與 SRF S99A 質體一同轉染至細胞中，會減緩 CK2 促進的 SRE 啟動子轉錄活性。為了探討 CK2 調控 SRE 的轉錄活性在神經保護作用裡扮演的角色，因此，將 CK2 蛋白表現量增加是否會保護 PC12 細胞對抗Rotenone所誘發的細胞凋亡傷害?結果顯示 CK2 表現量增加會保護細胞對抗Rotenone誘導的細胞凋亡，並減緩 Rotenone 對 SRE 調控的轉錄活性降低，但是，突變型 SRF S99A 蛋白會降低 CK2α 的影響作用。這些結果顯示 BDNF 促進 SRE 調控的基因表現是會透過 CK2 訊息傳遞路徑。

The neurotrophins play an important role in cell differentiation and survival of the nervous system. Among them, the neuroprotective effects of brain-derived neurotrophic factor (BDNF) is showed to be mediated by extracellular signal-regulated kinase (ERK) and phosphatidylinositol-3 kinase (PI3K) signaling pathway in the recent studies. However, other cellular signaling pathways might be involved in these effects of BDNF. Protein kinase CK2 (casein kinase 2) is a ubiquitous and highly conserved serine/threonine protein kinase and is indicated as a vital cellular role. In recent years, evidences have been mounted in support of the importance of CK2 in the suppression of apoptosis. Serum response factor (SRF) is a transcription factor binding to a consensus DNA sequence SRE (known as a CArG box) which was found in the promoters of some immediately early genes (such as c-fos, Egr) and anti-apoptotic Mcl-1 gene. The activations of SRF-regulated genes were associated with cell proliferation, cell survival and perception of synaptic activity. However, the regulatory mechanism of SRE-mediated genes is not well studied. The SRE-mediated transcription activity through CK2 signaling by BDNF treatment was studied in the PC12 cells in the present study. Results revealed that BDNF significantly increased the SRE promoter activity by luciferase report assay. The SRE-mediated transcription activity was increased by overexpression of CK2α, and the inhibition of endogenous CK2α by small interfering RNA was also shown to reduce this transcription activity. Furthermore, CK2α siRNA treatment antagonized the up-regulation effects of BDNF on SRE-mediated transcription activity. The co-transfection of CK2 and mutant SRF S99A plasmids significantly diminished up-regulatory effects of CK2 on SRE promoter activity. To test this CK2 induction in SRE-mediated transcription plays a role in neuroprotecion, we determined whether over-expression CK2 protects PC12 cells against rotenone-induced apoptosis. The results revealed that the over-expression of CK2α protected cells against rotenone-induced apoptosis and rescued the SRE-mediated transcription activity. Further, these effects of CK2α were blocked by co-transfection of mutant SRF S99A. These above results demonstrate that the up-regulation of BDNF on SRE-mediated genes is through CK2 signaling pathway.