TGFbeta Induces Binucleation/Polyploidization in Hepatocytes through a Src-Dependent Cytokinesis Failure

In all mammals, the adult liver shows binucleated as well as mononucleated polyploid hepatocytes. The hepatic polyploidization starts after birth with an extensive hepatocyte binucleation and generates hepatocytes of several ploidy classes. While the functional significance of hepatocyte polyploidy is becoming clearer, how it is triggered and maintained needs to be clarified. Aim of this study was to identify a major inducer of hepatocyte binucleation/polyploidization and the cellular and molecular mechanisms involved. We found that, among several cytokines analyzed, known to be involved in early liver development and/or mass control, TGFbeta1 was capable to induce, together with the expected morphological changes, binucleation in hepatocytes in culture. Most importantly, the pharmacological inhibition of TGFbeta signaling in healthy mice during weaning, when the physiological binucleation occurs, induced a significant decrease of hepatocyte binucleation rate, without affecting cell proliferation and hepatic index. The TGFbeta-induced hepatocyte binucleation resulted from a cytokinesis failure, as assessed by video microscopy, and is associated with a delocalization of the cytokinesis regulator RhoA-GTPase from the mid-body of dividing cells. The use of specific chemical inhibitors demonstrated that the observed events are Src-dependent. Finally, the restoration of a fully epithelial phenotype by TGFbeta withdrawal gave rise to a cell progeny capable to maintain the polyploid state. In conclusion, we identified TGFbeta as a major inducer of hepatocyte binucleation both in vitro and in vivo, thus ascribing a novel role to this pleiotropic cytokine. The production of binucleated/tetraploid hepatocytes is due to a cytokinesis failure controlled by the molecular axis TGFbeta/Src/RhoA.

在所有哺乳动物体内，成年肝脏中同时存在双核肝细胞（binucleated hepatocytes）与单核多倍体肝细胞（mononucleated polyploid hepatocytes）。肝脏多倍体化过程始于出生后，伴随广泛的肝细胞双核化事件，进而产生多种倍性类别的肝细胞。尽管肝细胞多倍体化的功能意义正逐步明晰，但其触发与维持的具体机制仍有待阐明。本研究旨在明确肝细胞双核化/多倍体化的主要诱导因子，以及其所涉及的细胞与分子机制。我们经分析发现，在多种已被证实参与肝脏早期发育和/或肝脏质量调控的细胞因子中，转化生长因子β1（TGFbeta1）可在体外培养的肝细胞中诱导双核化，同时伴随预期的形态学改变。尤为关键的是，在生理双核化发生的断奶阶段，对健康小鼠的TGFβ信号通路进行药物抑制，可显著降低肝细胞的双核化率，且不会影响细胞增殖与肝脏指数。通过视频显微镜观察验证，TGFβ诱导的肝细胞双核化源于胞质分裂（cytokinesis）失败，且该过程伴随胞质分裂调控因子RhoA鸟苷三磷酸酶（RhoA-GTPase）从分裂细胞的中体（mid-body）发生脱定位。使用特异性化学抑制剂开展的实验证实，上述事件依赖于Src通路。最后，撤除TGFβ以恢复完全上皮表型后，可获得能够维持多倍体状态的细胞子代。综上，我们在体外与体内实验中均确认TGFβ是肝细胞双核化的主要诱导因子，从而为这一多效性细胞因子赋予了全新的生物学功能。双核/四倍体肝细胞的产生，源于受TGFβ/Src/RhoA分子轴调控的胞质分裂失败。