於離乳前Wnt/ β-catenin訊息路徑對於調控肝細胞多套染色體化之功能角色

Abstract

在哺乳類肝細胞當中，多套染色體化(polyploidization)是有別於一般細胞之特殊現象，但其調控機制及重要性尚不清楚。在小鼠肝細胞中，多套染色體化是於離乳後開始，經由胞質分裂失敗(cytokinesis failure)而產生雙核四套的肝細胞(binucleated tetraploid hepatocytes)，而後套數伴隨年齡增加而緩慢上升。目前已知insulin/akt 訊息路徑及E2F8，對於肝細胞多套染色體化的調控中扮演促進因子的角色，在小鼠離乳後，insulin/akt 訊息路徑及E2F8表現上升，但其上游調控機制仍未知。有鑑於Wnt/β-catenin訊息路徑活化在小鼠離乳後下降，與已知兩個促進因子呈現相反的趨勢，本論文因此提出測試Wnt/β-catenin訊息路徑對於調控肝細胞多套染色體化可能為一負向調控角色之假說。我們首先利用免疫螢光染色法及DNA套數染色分析，確認在野生型小鼠離乳後肝細胞多套染色體化比例增加，此外並利用西方墨點法分析以確認在小鼠離乳後，Wnt/β-catenin訊息路徑活化的降低與 Akt 活化及E2F8表現上升之關聯性。 為了探究 Wnt/β-catenin訊息路徑是否在小鼠離乳前扮演抑制肝細胞多套染色體化的因子，我們利用白蛋白啟動子(albumin promoter)驅使Cre 重組蛋白(Cre recombinase)，在肝細胞特定(hepatocytes-specific)中於出生後剔除(postnatal knockout) β-catenin，觀察離乳前β-catenin剔除(β-catenin knockout, BKO)小鼠肝細胞中雙核比例的變化。發現相較於野生型(wildtype, WT)小鼠，BKO小鼠肝細胞雙核比例顯著上升，並且在β-catenin剔除後的肝細胞中，對於調控胞質分裂過程相關的基因表現確實被影響.，顯示Wnt/β-catenin訊息路徑可能扮演著抑制胞質分裂失敗的角色，維持肝細胞的正常胞質分裂。接著，我們想探討Wnt/β-catenin訊息路徑抑制胞質分裂失敗，其機制是否和目前已知insulin/akt 訊息路徑及E2F8兩個促進肝細胞多套染色體化的因子相關。我們利用西方墨點法分析，觀察離乳前BKO小鼠肝臟中，Akt訊息路徑活化以及目前已知與E2F8表現相反之E2F1的表現，發現相較於野生型小鼠，BKO小鼠肝臟中Akt訊息路徑活化上升且E2F1表現下降，顯示出Wnt/β-catenin訊息路徑抑制胞質分裂失敗之機制，可能是透過抑制Akt訊息路徑及E2F8此兩促進因子所造成，暗示著活化的β-catenin對於離乳前的肝臟，對於此兩正向調控因子為一個上游負向調控者。此外，我們發現在BKO小鼠肝細胞中PCNA增生能力降低，支持Wnt/β-catenin訊息路徑對於促進細胞增生能力扮演重要的角色。本篇論文的結論是，發現於離乳前肝細胞中Wnt/β-catenin 訊息路徑活化的功能，不只是刺激細胞增生，同時經由抑制Akt 及E2F8 路徑而確保成功的胞質分裂，其詳細機制以及對於人類肝臟相關疾病的涵義值得更進一步研究與探討。

Polyploidization is a specific feature in mammalian hepatocytes, but the underlying regulatory mechanism and its significance in liver biology is still remained inconclusive. In rodents, the hepatic polyploidization starts during weaning period, with the increase of binucleated tetraploid hepatocytes mainly caused by cytokinesis failure. Previous studies have identified the insulin/akt pathway and E2F8 as positive regulators for hepatic polyploidization, which are increased after weaning, but its upstream regulator is remained unidentified. It is noteworthy that Wnt/β-catenin activity after weaning in mice, in an opposite manner for these two positive regulators. Therefore, the current study proposed to test the hypothesis that if the Wnt/β-catenin pathway could be a negative regulator for hepatic polyploidization at weaning period, in antagonizing of the Akt and E2F8 pathways. By immunofluorescence staining and flow cytometry analysis, we have first validated the increase of polyploidy in hepatocytes after weaning in wild type mice. In addition, we have also verified the decreased Wnt/β-catenin activity, increased Akt activity, and decrease of E2F1 expression (a negative regulator for E2F8) after weaning. To investigate whether Wnt/β-catenin pathway inhibits hepatic polyploidization before weaning, we have tried to examine the effect of depleting β-catenin before weaning for the polidy changes. The hepatocyte specific β-catenin knockout mice (BKO mice), with the Cre expression driven by the albumin promoter, were established to knock out β-catenin in postnatal hepatocytes. We found that the proportion of hepatocytes containing binucleated tetraploidy in BKO mice is significantly increased compared to the wild type mice, and the cytokinesis-related genes are indeed affected in the BKO hepatocytes as expected. These results revealed that Wnt/β-catenin pathway plays a critical role in inhibiting the cytokinesis failure event to ensure the normal cytokinesis before weaning. Next, we have tried to examine if the Wnt/β-catenin pathway regulates the hepatic polyploidy through the insulin/Akt and E2F8 pathways. The results of western blot analysis revealed that the Akt activity is increased and the E2F1 expression is decreased in the BKO liver before weaning, suggesting the function of Wnt/β-catenin pathway in regulating the cytokinesis failure event through inhibiting the Akt and E2F8 pathways. Our study thus identified the active β-catenin pathway as a potential upstream negative regulator for these two positive regulators for hepatic polyploidization before weaning period in mice. Meanwhile, we found a decreased PCNA expression pattern in the hepatocytes of BKO mice, supporting the critical role of Wnt/β-catenin pathway in promoting hepatocyte proliferation before weaning. In summary, the active Wnt/β-catenin not only functions to stimulate cell proliferation but also at the same time ensure the successful cytokinesis in hepatocytes before the weaning period. The detailed mechanism and its implication for the liver diseases in adult liver are worthy to be further investigated.