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

ABSTRACT: 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 in silico 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.

SUBMITTER: Vig S

PROVIDER: S-EPMC4614533 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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

Vig Saurabh S Talwar Puneet P Kaur Kirandeep K Srivastava Rohit R Srivastava Arvind K AK Datta Malabika M

Cell cycle (Georgetown, Tex.) 20150506 14

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 in silico 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 leve ...[more]

PMID: 25946468

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Project description:Aims: To identify transcriptome signatures underlying epileptogenesis in temporal lobe epilepsy (TLE).Methods: Robust rank aggregation analysis was used to integrate multiple microarrays in rodent models of TLE and determine differentially expressed genes (DEGs) in acute, latent, and chronic stages. Functional annotation and protein-protein interaction analysis were performed to explore the potential functions of the DEGs and identify hub genes with the highest intramodular connectivity. The association between hub genes and hippocampal sclerosis/seizure frequency was analyzed using publicly available RNA-sequencing datasets from TLE patients. We subsequently established a pilocarpine-induced status epilepticus (SE) model in rats and validated mRNA expression of hub genes by quantitative reverse transcription PCR (qRT-PCR).Results: The DEGs in the acute, latent, and chronic phases of TLE in animal models were prominently enriched in inflammatory response. Hub genes identified in the acute phase mainly participated in biological processes including inflammation, blood-brain barrier damage, and cell adhesion. The hub genes in the latent phase were related to microglia/macrophage activation (Emr1 and Aif1) and phagocytosis (Cd68, Tyrobp, and Lyz). In the chronic phase, the hub genes were associated with activation of complements and microglia/macrophages. We further found that some hub genes identified in human TLE, such as Tlr2, Lgals3, and Stat3, were positively correlated with seizure frequency. Other hub genes, including Lgals3 and Serpine1, were associated with hippocampus sclerosis. qRT-PCR analysis confirmed that the mRNA levels of hub genes in rat hippocampus were significantly up-regulated after SE induction.Conclusions: Our integrated analysis identified hub genes in different stages of epilepsy. The functional annotations suggest that the activation and phagocytic activities of microglia/macrophages may play critical roles in epileptogenesis of TLE.

Project description:BackgroundEthanol abuse promotes breast cancer development, metastasis and recurrence stimulating mammary tumorigenesis by mechanisms that remain unclear. Normally, 35% of breast cancer is Erb-B2 Receptor Tyrosine Kinase 2 (ERBB2)-positive that predisposes to poor prognosis and relapse, while ethanol drinking leads to invasion of their ERBB2 positive cells triggering the phosphorylation status of mitogen-activated protein kinase. StAR-related lipid transfer protein 10 (STARD10) is a lipid transporter of phosphatidylcholine (PC) and phosphatidylethanolamine (PE); changes on membrane composition of PC and PE occur before the morphological tumorigenic events. Interestingly, STARD10 has been described to be highly expressed in 35-40% of ERBB2-positive breast cancers. In this study, we demonstrate that ethanol administration promotes STARD10 and ERBB2 expression that is significantly associated with increased cell malignancy and aggressiveness.Material and methodsWe investigated the effect of ethanol on STARD10-ERBB2 cross-talk in breast cancer cells, MMTV-neu transgenic mice and in clinical ERBB2-positive breast cancer specimens with Western Blotting and Real-time PCR. We also examined the effects of their knockdown and overexpression on transient transfected breast cancer cells using promoter activity, MTT, cell migration, calcium and membrane fluidity assays in vitro.ResultsEthanol administration induces STARD10 and ERBB2 expression in vitro and in vivo. ERBB2 overexpression causes an increase in STARD10 expression, while overexpression of ERBB2's downstream targets, p65, c-MYC, c-FOS or c-JUN induces STARD10 promoter activity, correlative of enhanced ERBB2 function. Ethanol and STARD10-mediated cellular membrane fluidity and intracellular calcium concentration impact ERBB2 signaling pathway as evaluated by enhanced p65 nuclear translocation and binding to both ERBB2 and STARD10 promoters.ConclusionOur finding proved that STARD10 and ERBB2 positively regulate each other's expression and function. Taken together, our data demonstrate that ethanol can modulate ERBB2's function in breast cancer via a novel interplay with STARD10.

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

Publications

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

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