Background & Aims

Hepatic stellate cells (HSCs) contribute to desmoplasia and stiffness of liver metastases by differentiating into matrix-producing myofibroblasts. We investigated whether stiffness due to the presence of tumors increases activation of HSCs into myofibroblasts and their tumor-promoting effects, as well as the role of E1A binding protein p300, a histone acetyltransferase that regulates transcription, in these processes.

Methods

HSCs were isolated from liver tissues of patients, mice in which the p300 gene was flanked by 2 loxP sites (p300F/F mice), and p300+/+ mice (controls). The HSCs were placed on polyacrylamide gels with precisely defined stiffness, and their activation (differentiation into myofibroblasts) was assessed by immunofluorescence and immunoblot analyses for alpha-smooth muscle actin. In HSCs from mice, the p300 gene was disrupted by cre recombinase. In human HSCs, levels of p300 were knocked down with small hairpin RNAs or a mutant form of p300 that is not phosphorylated by AKT (p300S1834A) was overexpressed. Human HSCs were also cultured with inhibitors of p300 (C646), PI3K signaling to AKT (LY294002), or RHOA (C3 transferase) and effects on stiffness-induced activation were measured. RNA sequencing and chromatin immunoprecipitation–quantitative polymerase chain reaction were used to identify HSC genes that changed expression levels in response to stiffness. We measured effects of HSC-conditioned media on proliferation of HT29 colon cancer cells and growth of tumors following subcutaneous injection of these cells into mice. MC38 colon cancer cells were injected into portal veins of p300F/Fcre and control mice, and liver metastases were measured. p300F/Fcre and control mice were given intraperitoneal injections of CCl₄ to induce liver fibrosis. Liver tissues were collected and analyzed by immunofluorescence, immunoblot, and histology.

Results

Substrate stiffness was sufficient to activate HSCs, leading to nuclear accumulation of p300. Disrupting p300 level or activity blocked stiffness-induced activation of HSCs. In HSCs, substrate stiffness activated AKT signaling via RHOA to induce phosphorylation of p300 at serine 1834; this caused p300 to translocate to the nucleus, where it up-regulated transcription of genes that increase activation of HSCs and metastasis, including CXCL12. MC38 cells, injected into portal veins, formed fewer metastases in livers of p300F/Fcre mice than control mice. Expression of p300 was increased in livers of mice following injection of CCl4; HSC activation and collagen deposition were reduced in livers of p300F/Fcre mice compared with control mice.

Conclusions

In studies of mice, we found liver stiffness to activate HSC differentiation into myofibroblasts, which required nuclear accumulation of p300. p300 increases HSC expression of genes that promote metastasis.

中文翻译：

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P300乙酰转移酶介导刚度诱导的肝星状细胞激活进入促肿瘤成肌纤维细胞。

背景与目标

肝星状细胞（HSC）通过分化为可产生基质的成肌纤维母细胞，促进了肝转移的增生和僵硬。我们调查了由于肿瘤的存在而引起的僵硬在这些过程中是否增加了HSCs向成肌纤维细胞的激活及其促肿瘤作用，以及E1A结合蛋白p300（一种调节转录的组蛋白乙酰转移酶）的作用。

方法

从患者的肝组织，p300基因两侧有2个loxP位点的小鼠（p300 F / F小鼠）和p300 +中分离出HSC/ +小鼠（对照）。将HSCs以精确定义的刚度放置在聚丙烯酰胺凝胶上，并通过免疫荧光和免疫印迹分析α-平滑肌肌动蛋白评估其活化（分化为成肌纤维细胞）。在小鼠的HSC中，cre重组酶破坏了p300基因。在人类HSC中，p300的水平被小发夹RNA击倒，或者未被AKT磷酸化的p300突变形式（p300S1834A）过表达。还用p300（C646），向AKT的PI3K信号转导（LY294002）或RHOA（C3转移酶）的抑制剂培养人类HSC，并测量其对僵硬诱导的激活的影响。RNA测序和染色质免疫沉淀-定量聚合酶链反应用于鉴定HSC基因，这些基因可改变表达水平以响应刚度。我们测量了HSC条件培养基对HT29结肠癌细胞增殖和皮下注射这些细胞到小鼠体内后肿瘤生长的影响。将MC38结肠癌细胞注入门静脉测量p300 F / F cre和对照组小鼠的肝转移情况。对p300 F / F cre和对照组小鼠进行腹腔注射CCl ₄诱导肝纤维化。收集肝组织并通过免疫荧光，免疫印迹和组织学分析。

结果

底物刚度足以激活HSC，导致p300的核积累。破坏p300的水平或活动可阻止刚度诱导的HSC激活。在HSC中，底物刚度通过RHOA激活了AKT信号传导，从而在1834年的丝氨酸上诱导了p300的磷酸化。这导致p300易位至细胞核，从而上调了增加HSC激活和转移的基因（包括CXCL12）的转录。与对照小鼠相比，注入门静脉的MC38细胞在p300F / F cre小鼠的肝脏中形成的转移较少。注射CCl4后，小鼠肝脏中p300的表达增加；与对照小鼠相比，p300 F / F cre小鼠肝脏中的HSC活化和胶原沉积减少。

结论

在对小鼠的研究中，我们发现肝刚度可以激活HSC分化为成肌纤维细胞，这需要p300的核积累。p300增加促进转移的基因的HSC表达。