Vps33b is crucial for structural and functional hepatocyte polarity.
Vps33b is crucial for structural and functional hepatocyte polarity.
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DOI：
10.1016/j.jhep.2017.01.001
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发表时间：
2017-05
影响因子：
25.7
通讯作者：
Gissen P
中科院分区：
文献类型：
--
作者：
Hanley J;Dhar DK;Mazzacuva F;Fiadeiro R;Burden JJ;Lyne AM;Smith H;Straatman-Iwanowska A;Banushi B;Virasami A;Mills K;Lemaigre FP;Knisely AS;Howe S;Sebire N;Waddington SN;Paulusma CC;Clayton P;Gissen P
在正常肝脏中，肝细胞形成独特的极化细胞层，使溶质能够从窦状血移动到小管胆汁。虽然已经鉴定了几种具有肝细胞极性缺陷的胆汁淤积性肝病，但发病机制的分子机制尚未明确。一个例子是关节弯曲，肾功能不全和胆汁淤积综合征，这在大多数患者中是由VPS 33 B突变引起的。VPS 33 B是参与膜运输的蛋白质，其在再循环内体处与RAB 11 A相互作用。为了更好地了解调节肝细胞极性的途径，我们使用具有肝脏特异性Vps 33 b缺失的新型小鼠模型研究了VPS 33 B缺陷。为了评估功能极性，从Vps 33 b肝敲除（Vps 33 bfl/fl-AlfpCre）和对照（Vps 33 bfl/fl）小鼠收集血浆和胆汁样品;通过质谱法或荧光测定法定量胆汁组分或注射的底物。对于结构分析，肝脏进行了光学和透射电子显微镜检查。顶膜和紧密连接蛋白定位进行了评估，免疫染色。腺相关病毒载体用于体内基因拯救实验。与患者一样，Vps 33 bfl/fl-AlfpCre小鼠显示ATP结合盒蛋白的错误定位，这些蛋白通过Rab 11 a阳性再循环内体特异性地运输到顶端膜。这与胆汁成分在血液中的滞留有关。在基因敲除动物中，观察到功能性紧密连接完整性的丧失和顶端微绒毛的耗竭。基因转移部分挽救了这些缺陷。Vps 33 b在建立肝细胞极性的结构和功能方面具有关键作用，并且可能是基因替代疗法的靶点。肝细胞是具有顶部和底部的肝细胞;也就是说，它们是极化的。在它们的底部，它们从血液中吸收物质。然后，在它们的顶部，它们将这些物质及其代谢物分泌到胆汁中。当极性丧失时，这种物质从血液到胆汁的定向流动被破坏，肝脏疾病随之而来。在这项研究中，使用一种新的小鼠模型与肝脏特异性突变的Vps 33 b，小鼠版本的基因突变，在大多数患者关节弯曲，肾功能不全和胆汁淤积（ARC）综合征，我们调查了如何Vps 33 b基因产物有助于建立肝细胞极性。我们在这些小鼠中发现了与ARC综合征儿童相似的异常。基因转移可以部分逆转小鼠的异常。我们的工作有助于理解VPS 33 B疾病和肝细胞极性，并可能指向基因转移介导的ARC肝病治疗。
In the normal liver, hepatocytes form a uniquely polarised cell layer that enables movement of solutes from sinusoidal blood to canalicular bile. Whilst several cholestatic liver diseases with defects of hepatocyte polarity have been identified, the molecular mechanisms of pathogenesis are not well defined. One example is arthrogryposis, renal dysfunction and cholestasis syndrome, which in most patients is caused by VPS33B mutations. VPS33B is a protein involved in membrane trafficking that interacts with RAB11A at recycling endosomes. To understand the pathways that regulate hepatocyte polarity better, we investigated VPS33B deficiency using a novel mouse model with a liver-specific Vps33b deletion. To assess functional polarity, plasma and bile samples were collected from Vps33b liver knockout (Vps33bfl/fl-AlfpCre) and control (Vps33bfl/fl) mice; bile components or injected substrates were quantitated by mass spectrometry or fluorometry. For structural analysis, livers underwent light and transmission electron microscopy. Apical membrane and tight junction protein localisation was assessed by immunostaining. Adeno-associated virus vectors were used for in vivo gene rescue experiments. Like patients, Vps33bfl/fl-AlfpCre mice showed mislocalisation of ATP-binding cassette proteins that are specifically trafficked to the apical membrane via Rab11a-positive recycling endosomes. This was associated with retention of bile components in blood. Loss of functional tight junction integrity and depletion of apical microvilli were seen in knockout animals. Gene transfer partially rescued these defects. Vps33b has a key role in establishing structural and functional aspects of hepatocyte polarity and may be a target for gene replacement therapy. Hepatocytes are liver cells with tops and bottoms; that is, they are polarised. At their bottoms they absorb substances from blood. They then, at their tops, secrete these substances and their metabolites into bile. When polarity is lost, this directional flow of substances from blood to bile is disrupted and liver disease follows. In this study, using a new mouse model with a liver-specific mutation of Vps33b, the mouse version of a gene that is mutated in most patients with arthrogryposis, renal dysfunction and cholestasis (ARC) syndrome, we investigated how the Vps33b gene product contributes to establishing hepatocyte polarity. We identified in these mice abnormalities similar to those in children with ARC syndrome. Gene transfer could partly reverse the mouse abnormalities. Our work contributes to the understanding of VPS33B disease and hepatocyte polarity in general, and may point towards gene transfer mediated treatment of ARC liver disease.