鋰鹽藉減少轉錄因子Elf3 mRNA降低第二型水通道蛋白質的表現

Abstract

抗利尿激素(vasopressin(AVP))是一種多胜肽賀爾蒙，當其從腦下垂體釋放跟著血流來到腎臟，會增加集尿管細胞對水的通透性，增加水分再吸收回體內，而降低水份從尿液中排除。AVP調整細胞對水的通透性，主要透過短期的調控水通道蛋白質2號(aquaporin-2(AQP2))的運輸，和長期的調控AQP2的基因表現。當AVP無法正常誘導AQP2基因表現時，會造成一些與水分平衡失調有關的疾病，其中包括鋰鹽誘導的腎源性尿崩症(lithium-induced nephrogenic diabetes insipidus)，但其分子機制尚未明瞭。先前實驗室發現一個轉錄因子Elf3參與在AVP所誘導的AQP2基因表現的機制當中。我的研究目標是鋰鹽是否透過減少Elf3 mRNA的表現量而導致AQP2的表現量下降。免疫螢光染色的結果顯示，在AVP類似物dDAVP的刺激下，會促進帶有V5-tag的Elf3進到細胞核內。在即時聚合酶連鎖反應的分析下，40mM的鋰鹽會造成Elf3的mRNA減少並伴隨著減少AQP2的mRNA，而且不影響細胞生存。在西方點墨法中也發現40mM的鋰鹽會使AQP2 protein表現量減少，而上述的影響並非鋰鹽的滲透壓所導致，因為使用相同滲透壓的鈉鹽並沒有產生相同的結果。這些實驗表示鋰鹽抑制AVP所誘導的AQP2基因表現可能是透過降低Elf3的mRNA所導致。在分子機制方面，我們發現鋰鹽會增加醣原合成激酶(glycogen synthase kinase 3β(GSK3β))上絲氨酸9號(serine 9)的磷酸化，而抑制GSK3β。GSK3β的下游目標之一是nuclear factor-κB (NF-κB)，且NF-κB在Elf3的啟動子區有結合位。因此，鋰鹽可能透過影響NF-κB而減少Elf3轉錄。然而，knockdown NF-κB並沒有影響到Elf3 mRNA表現量，這表示鋰鹽降低Elf3 mRNA可能不是藉由NF-κB調控。有趣的是knockdown NF-κB卻顯著地降低AVP所誘導的AQP2表現。我們的結論是鋰鹽誘導的尿崩症可能是透過降低Elf3轉錄而減少Elf3所調控的AQP2基因表現所致。另外，NF-κB可能在AQP2基因表現上有直接的調控作用。

Vasopressin (AVP) is an antidiuretic peptide hormone that is released from the pituitary and circulates to the kidneys where it increases water permeability of the collecting ducts and reduces water excretion. AVP regulates the water permeability via short term regulation of aquaporin-2 (AQP2) trafficking and long term regulation of AQP2 gene expression. Dysregulation of vasopressin-mediated long term AQP2 gene expression is associated with many water balance diseases including lithium-induced nephrogenic diabetes insipidus; however, the molecular mechanism of the pathogenesis remains unclear. Previously, our laboratory found that the transcription factor Elf3 mediates vasopressin-induced AQP2 gene expression. The aim of my study is to investigate whether lithium down-regulates vasopressin-induced AQP2 gene expression via affecting the expression of Elf3 that has a binding site in the AQP2 promoter region. Immunofluorescence staining showed that the vasopressin analog dDAVP stimulated V5-tagged Elf3 translocation into the nucleus of the collecting duct cell model mpkCCD. Quantitative RT-PCR analysis showed that 40mM lithium treatment was enough to decrease Elf3 mRNA and vasopressin-induced increases in the AQP2 mRNA level in the mpkCCD cells without affecting cell viability measured with the MTT assay. Immunoblotting showed that 40mM lithium reduced vasopressin-induced increases in the AQP2 protein levels. The above effects were not results of the lithium tonicity because sodium at the same tonicity did not produce the same results. These data suggest that lithium suppresses vasopressin-induced AQP2 gene expression via reducing Elf3 mRNA. To investigate the molecular mechanism involved, we found that lithium increased phosphorylation at serine 9 of glycogen synthase kinase 3β (GSK3β), a well-known target of lithium. One of the downstream targets of GSK3β is nuclear factor-κB (NF-κB) that has a binding site on the Elf3 promoter region. This led to the hypothesis that lithium could potentially affect NF-κB to reduce Elf3 transcription. However, NF-κB knockdown did not affect the Elf3 mRNA levels, suggesting that the lithium-induced decrease in the Elf3 mRNA level is probably not mediated by NF-κB. One interesting finding was that NF-κB knockdown significantly reduced vasopressin-induced increased in the AQP2 mRNA levels. In summary, we conclude that lithium-induced diabetes insipidus is probably due to a reduced transactivation activity of Elf3 that mediates vasopressin-induced AQP2 gene expression and that NF-κB probably plays a direct role in vasopressin-induced AQP2 gene expression.