Dexamethasone促進抗利尿激素誘導小鼠集尿管細胞表現第二型水通道基因

Abstract

第二型水通道蛋白質(AQP2)表現於腎臟集尿管中，並且參與腎臟的水分再吸收作用。AQP2的基因表現會受到抗利尿激素(arginine vasopressin (AVP))的調控，但其分子機制尚不清楚。為了進一步研究調控AQP2的分子機制，科學家嘗試建立細胞株作為研究平台。但可能因為建立的過程使細胞喪失集尿管細胞的特性，因此許多細胞株都無法對AVP的刺激產生反應並表現內生性的AQP2。這個問題直到Vandewalle的團隊用「Trans-immortalization」的方式建立小鼠集尿管細胞株(mpkCCD)後才獲得解決。mpkCCD藉由攜帶一段在丙酮酸激酶(LPK)的啟動子後方接上SV40 large T抗原基因的DNA片段，使細胞穩定表現T抗原，藉此幫助細胞維持生長的能力。科學家也透過在培養液中加入dexamethasone (Dex)、表皮生長因子、胰島素及甲狀腺素等賀爾蒙來幫助活化LPK啟動子，以維持T抗原的表現。這些賀爾蒙也曾被報導對AQP2基因表現可能有不同的影響，因此在這份研究中，我針對Dex、表皮生長因子、胰島素及甲狀腺素是否影響mpkCCD細胞中AQP2的表現進行探討。我發現Dex會提升AVP所誘導的AQP2蛋白質表現量，而此增加並非透過影響AQP2的蛋白穩定性。此外，AQP2在Dex處理之下所提升的蛋白表現量及mRNA表現量有相似的趨勢，此增加也伴隨著AQP2啟動子活性的上升。上述結果顯示Dex可能是透過促進AQP2的轉錄作用，進而提升AQP2 mRNA及蛋白質的表現量。在分子機制方面，我們發現Dex會增加抗利尿激素受體(V2R) mRNA的表現量，顯示Dex可能透過提升V2R mRNA表現來增加細胞對AVP的反應。除此之外，我們也發現Dex提升了Elf3 (一種被認為參與AQP2調控的轉錄因子) mRNA表現量，針對Elf3在AQP2啟動子上的結合位(ETS-binding site)進行突變也降低了Dex對AVP所誘導的AQP2啟動子活性的影響，顯示Dex可能透過增加Elf3 mRNA表現量來調節AVP所誘導的AQP2基因表現。從上述的實驗我們發現了一個能夠提升mpkCCD細胞表現AQP2能力的條件，希望能夠幫助AVP和AQP2相關研究領域的發展。

Aquaporin-2 (AQP2) is a molecular channel protein responsible for osmotic water reabsorption by the kidneys. AQP2 gene expression is tightly regulated by the pituitary peptide hormone called arginine vasopressin (AVP); however, the molecular mechanism is largely unknown. Research in this field has been slow due to the lack of proper cell lines that respond to AVP with endogenous AQP2 expression until the development of the mpkCCD cells. The mpkCCD cells were immortalized by the SV40 large T antigen under the L-type pyruvate kinase promoter. Four hormones (dexamethasone (Dex), epidermal growth factor, insulin and triiodothyronine) were used to drive the LPK promoter and promote cell growth; however, each seems to affect the levels of AVP-induced AQP2 expression. In the present study, we found that Dex, among all hormone supplements, enhanced AVP-induced AQP2 protein expression in a dose- and time-dependent manner without affecting AQP2 protein stability. This increase was accompanied with an increase in the AQP2 promoter activity and AVP-induced AQP2 mRNA levels, indicating that Dex promotes AVP-induced AQP2 protein expression via enhancing AQP2 mRNA transcription. Furthermore, we found that Dex slightly increased the mRNA levels of AVP type 2 receptor (V2R), suggesting that Dex may enhance the cell responses to AVP via increasing V2R mRNA expression. Moreover, Dex also increased the mRNA levels of Elf3, a collecting duct cell-specific transcription factor that binds the ETS-binding site in the AQP2 promoter region. Mutation in the ETS-binding site reduced the effects of Dex on AVP-induced AQP2 promoter activity. These results suggest that Dex probably promotes AQP2 gene expression through enhancing Elf3 gene expression. In summary, we have found an optimal condition to enhance AVP-induced AQP2 gene expression in the mpkCCD and hopefully this finding will facilitate AVP and AQP2 research.