Transcriptome profiling identifies p53 as a key player during calreticulin deficiency: Implications in lipid accumulation

Calreticulin (CRT) is an endoplasmic reticulum (ER) resident calcium binding protein that is involved in several cellular activities. Transcriptome analyses in CRT knockdown HepG2 cells revealed 253 altered unique genes and subsequent <i>in silico</i> protein-protein interaction network and MCODE clustering identified 34 significant clusters, of which p53 occupied the central hub node in the highest node-rich cluster. Toward validation, we show that CRT knockdown leads to inhibition of p53 protein levels. Both, CRT and p53 siRNA promote hepatic lipid accumulation and this was accompanied by elevated SREBP-1c and FAS levels. p53 was identified to bind at −219 bp on the SREBP-1c promoter and in the presence of CRT siRNA, there was decreased occupancy of p53 on this binding element. This was associated with increased SREBP-1c promoter activity and both, mutation in this binding site or p53 over-expression antagonised the effects of CRT knockdown. We, therefore, identify a negatively regulating p53 binding site on the SREBP-1c promoter that is critical during hepatic lipid accumulation. These results were validated in mouse primary hepatocytes and toward a physiological relevance, we report that while the levels of CRT and p53 are reduced in the fatty livers of diabetic db/db mice, SREBP-1c levels are significantly elevated. Our results suggest that decreased CRT levels might be involved in the development of a fatty liver by preventing p53 occupancy on the SREBP-1c promoter and thereby facilitating SREBP-1c up-regulation and consequently, lipid accumulation.

钙网蛋白（Calreticulin，CRT）是一种内质网（endoplasmic reticulum，ER）驻留的钙结合蛋白，参与诸多细胞生理活动。对CRT敲低的HepG2细胞进行转录组分析，共鉴定出253个差异表达的特异性基因；后续通过计算机模拟（in silico）蛋白质相互作用网络分析及MCODE聚类，识别出34个显著聚类模块，其中p53在节点丰度最高的聚类模块中占据核心枢纽节点位置。为验证上述结论，本研究证实CRT敲低会抑制p53蛋白的表达水平。CRT与p53小干扰RNA（small interfering RNA，siRNA）均可促进肝脏脂质蓄积，同时伴随固醇调节元件结合蛋白1c（SREBP-1c）及脂肪酸合酶（Fatty Acid Synthase，FAS）表达水平升高。研究发现p53可结合于SREBP-1c启动子的−219 bp位点；当使用CRT siRNA处理时，p53在该结合元件上的富集水平显著降低。这一现象与SREBP-1c启动子活性升高相关；且该结合位点突变或p53过表达均可拮抗CRT敲低所产生的上述效应。因此，本研究在SREBP-1c启动子上鉴定出一个负调控性p53结合位点，该位点在肝脏脂质蓄积过程中发挥关键作用。本研究在小鼠原代肝细胞中验证了上述结果；为探究其生理相关性，本研究发现：在糖尿病db/db小鼠的脂肪肝肝脏中，CRT与p53的表达水平均出现降低，而SREBP-1c的表达水平则显著升高。本研究结果表明，CRT表达水平降低可通过阻碍p53结合至SREBP-1c启动子，进而促进SREBP-1c的上调表达，最终导致肝脏脂质蓄积，参与脂肪肝的发生发展过程。