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標題: HK-2細胞在面臨高滲透壓環境時,其FXYD2 isoform 3在調控Na+, K+-ATPase活性上所扮演的可能角色
The potential role of FXYD2 isoform 3 in regulation of Na+, K+-ATPase activity in HK-2 cells upon hypertonic challenge
作者: 賴馨慈
Lai, Hsing-Tzu
關鍵字: hypertonic
FXYD2 isoform 3
NKA activity
出版社: 生命科學系所
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摘要: Na+, K+-ATPase (NKA)為廣泛存在細胞內且對於滲透壓調節極具重要性的運輸蛋白,其利用水解ATP排出三個鈉離子和吸收兩個鉀離子製造出離子濃度梯度,藉此造成其他二級主動運輸蛋白的驅動力。細胞利用調節NKA的功能來達到細胞對外界環境滲透壓變化的適應。哺乳類腎臟為扮演滲透壓平衡的調節器官,其中近曲小管為主要部位之一。因此本研究利用人類腎臟近曲小管表皮細胞(HK-2)給予高滲透壓刺激再搭配microarray的分析,篩選出與調節NKA功能相關且具高度表現之基因。其中FXYD2,一個具腎臟組織特異性且可影響NKA對鈉鉀親和力的蛋白質即為首選。在驗證microarray的結果時發現,FXYD2具有三個isoforms,其中isoform 3具有相對的顯著性表現。此外,isoform 3至今未有文獻探討其在滲透壓調節的功能,因此FXYD2 isoform 3為主要探討目標。實驗結果發現FXYD2 isoform 3和NKA α1 subunit的mRNA及蛋白表現量在高滲透壓下都有顯著性上升。另外也觀察到FXYD2 isoform 3的表現位置位於細胞膜上,而前人的研究也指出NKA α1 subunit表現在細胞膜上,所以更進一步去檢視FXYD2 isoform 3是否有跟NKA α1 subunit有交互作用,結果使用共同免疫沉澱得到他們確實有關係。為更深入探討FXYD2 isoform 3的角色,本研究使用了RNAi-A (knockdown isoform 1, 2, and 3)、RNAi-B (knockdown isoform 1 and 2)之細胞。FXYD2 isoforms 的knockdowm並未影響NKA α1 subunit的蛋白質表現,但NKA活性卻受到改變,NKA活性的排序由高至低為RNAi-B > RNAi-A > wild type。經由互相比較發現FXYD2 isoform 1, 2扮演著抑制NKA活性的角色;而FXYD2 isoform 3則是扮演促進NKA活性之角色。另一方面,在高滲透壓環境下,有文獻指出TonEBP為大量表現的轉錄因子,其主要活化其下游基因表現以保護細胞能適應此逆境並得以生存,所以TonEBP的調節機制對於細胞適應高滲透壓有舉足輕重的角色。經TonEBP knockdown實驗發現, TonEBP會影響FXYD2 isoform 3的mRNA和蛋白質表現,間接影響NKA的活性。綜合以上的結果本篇研究首先發現,在高滲透壓環境下HK-2細胞藉由TonEBP增加 FXYD2 isoform 3的表現,進而調節NKA之活性。
Na+,K+-ATPase (NKA) is a widely existence and important transporter in animal cells. NKA uptake two of K+ and extrusion of Na+ in providing the driving force to trigger the secondary active transporters by using the energy of the hydrolysis of ATP. Cells regulated NAK function to adapt the environmental osmotic changes. The mammalian kidney was the key organ in osmotic homeostasis and proximal tubule was the one of major tissue. This study used human renal proximal tubule cells HK-2 and microarray screened the up-regulated genes which correlate to NKA function upon hypertonic challenge. FXYD2 protein was specificity in kidney, and it could effect on sodium/potassium affinity of NKA. To confirm the microarray data, FXYD2 isoform 3 was markedly expressed. There were still no reporters to discuss detail function of isoform 3 in osmotic regulation. Therefore, FXYD2 isoform 3 was the target of this study with functional assay. In this study, the mRNA and protein abundance of FXYD2 isoform 3 were significantly increased upon hypertonic challenge. In addition, the localization of FXYD2 isoform 3 was distributed on cell membrane as NKA α1 subunit. Moreover, we obtained FXYD2 isoform 3 indeed had interaction with NKA α1 subunit. Accordingly, it was suggested that FXYD2 isoforms3 played the role in regulation of NKA activity. Therefore, the potential role of FXYD2 isoform 3 will be illustrated during hypertonic challenge by using the RNAi-A (knockdown isoform 1, 2 and 3) and RNAi-B (knockdown isoform 1 and 2) knockdown cells. FXYD2 knockdown cells did not affect NKA protein expression level, but NKA activity was affected upon hypertonic stress. NKA activity high to low was RNAi-B > RNAi-A > wild type. FXYD2 isoform 1and 2 played the repressor role, and isoform 3 played an enhancer role in NKA activity regulation. On the other hand, some studies indicated TonEBP was a transcription factor and it was higher expression upon hypertonic challenge. TonEBP modulated its downstream genes to protect cells could adapt the effects of hypertonic challenge. Hence, modulation mechanism of TonEBP was important when cells were faced hypertonic environment. In TonEBP knockdown cells, FXYD2 isoform 3 expression was regulated by TonEBP and result in affecting NKA activity. Take together, this study was the first reporter to demonstrate that hypertonicity promoted FXYD2 isoform 3 expression through TonEBP-dependent regulation, and then enhanced NKA activity in HK-2 cells.
其他識別: U0005-2707201102283100
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