Contribution of Elevated Glucose and Oxidized LDL to Macrophage Inflammation: A Role for PRAS40/Akt-Dependent Shedding of Soluble CD14.

Atherosclerosis, a process in which macrophages play a key role, is accelerated in diabetes. Elevated concentrations of serum-oxidized low-density lipoproteins (oxLDL) represent a common feature of both conditions. The main goal of this study was to determine the contribution of oxLDL to the inflammatory response of macrophages exposed to diabetic-mimicking conditions. THP1 cells and peripheral blood monocytes purified from non-diabetic healthy donors were cultured under normal (5 mM) or high glucose (HG) (15 mM) with oxLDL. Then, foam cell formation, expression of CD80, HLADR, CD23, CD206, and CD163, as well as toll-like receptor 4 (TLR4) and co-receptors CD36 and CD14 (both at the cell surface and soluble (sCD14)), and inflammatory mediators' production were measured by flow cytometry, RT-qPCR, or ELISA. Additionally, serum sCD14 was determined in subjects with subclinical atherosclerosis with and without diabetes by ELISA. Our results showed that oxLDL-mediated intracellular lipid accumulation via CD36 increased under HG and that HG + oxLDL enhanced TNF, IL1B, and IL8, and decreased IL10. Moreover, TLR4 was upregulated in macrophages under HG and monocytes of subjects with diabetes and atherosclerosis. Interestingly, HG-oxLDL upregulated CD14 gene expression, although its total cellular protein abundance remained unaltered. sCD14 shedding via PRAS40/Akt-dependent mechanisms, with pro-inflammatory activity, was significantly increased in cultured macrophages and plasma from subjects with diabetes and subclinical atherosclerosis or hypercholesterolemia. Our data support an enhanced synergistic pro-inflammatory effect induced by HG and oxLDL in cultured human macrophages, possibly explained by increased sCD14 shedding.

Epigenetic footprint enables molecular risk stratification of hepatoblastoma with clinical implications.

BACKGROUND & AIMS: Hepatoblastoma (HB) is a rare disease. Nevertheless, it is the predominant pediatric liver cancer, with limited therapeutic options for patients with aggressive tumors. Herein, we aimed to uncover the mechanisms of HB pathobiology and to identify new biomarkers and therapeutic targets in a move towards precision medicine for patients with advanced HB. METHODS: We performed a comprehensive genomic, transcriptomic and epigenomic characterization of 159 clinically annotated samples from 113 patients with HB, using high-throughput technologies. RESULTS: We discovered a widespread epigenetic footprint of HB that includes hyperediting of the tumor suppressor BLCAP concomitant with a genome-wide dysregulation of RNA editing and the overexpression of mainly non-coding genes of the oncogenic 14q32 DLK1-DIO3 locus. By unsupervised analysis, we identified 2 epigenomic clusters (Epi-CA, Epi-CB) with distinct degrees of DNA hypomethylation and CpG island hypermethylation that are associated with the C1/C2/C2B transcriptomic subtypes. Based on these findings, we defined the first molecular risk stratification of HB (MRS-HB), which encompasses 3 main prognostic categories and improves the current clinical risk stratification approach. The MRS-3 category (28%), defined by strong 14q32 locus expression and Epi-CB methylation features, was characterized by CTNNB1 and NFE2L2 mutations, a progenitor-like phenotype and clinical aggressiveness. Finally, we identified choline kinase alpha as a promising therapeutic target for intermediate and high-risk HBs, as its inhibition in HB cell lines and patient-derived xenografts strongly abrogated tumor growth. CONCLUSIONS: These findings provide a detailed insight into the molecular features of HB and could be used to improve current clinical stratification approaches and to develop treatments for patients with HB. LAY SUMMARY: Hepatoblastoma is a rare childhood liver cancer that has been understudied. We have used cutting-edge technologies to expand our molecular knowledge of this cancer. Our biological findings can be used to improve clinical management and pave the way for the development of novel therapies for this cancer.

Increased Global DNA Hypomethylation in Distant Metastatic and Dedifferentiated Thyroid Cancer.

Context: Global DNA hypomethylation is a major event for the development and progression of cancer, although the significance in thyroid cancer remains unclear. Therefore, we aimed to investigate its role in thyroid cancer progression and its potential as a prognostic marker. Methods: Global hypomethylation of Alu repeats was used as a surrogate marker for DNA global hypomethylation, and was assessed using the Quantification of Unmethylated Alu technique. Mutations in BRAF and RAS were determined by Sanger sequencing. Results: Ninety primary thyroid tumors were included [28 low-risk differentiated thyroid cancer (DTC), 13 pediatric DTC, 33 distant metastatic DTC, 7 poorly differentiated thyroid cancer (PDTC), and 9 anaplastic thyroid cancer (ATC)], as well as 24 distant metastases and 20 normal thyroid tissues. An increasing hypomethylation was found for distant metastatic DTC [median, 4.0; interquartile range (IQR), 3.1 to 6.2] and PDTC/ATC (median, 9.3; IQR, 7.0 to 12.1) as compared with normal thyroid tissue (median, 2.75; IQR, 2.30 to 3.15), whereas low-risk and pediatric DTC were not affected by hypomethylation. Alu hypomethylation was similar between distant metastases and matched primary tumors. Within distant metastatic DTC, Alu hypomethylation was increased in BRAF vs RAS mutated tumors. Kaplan-Meier and Cox regression analyses showed that thyroid cancer-related and all-cause mortality were associated with tumor hypomethylation, but this association was lost after adjustment for thyroid cancer risk category. Conclusion: Distant metastatic DTC, PDTC, and ATC were increasingly affected by global Alu hypomethylation, suggesting that this epigenetic entity may be involved in thyroid cancer progression and dedifferentiation.

Altered CSNK1E, FABP4 and NEFH protein levels in the dorsolateral prefrontal cortex in schizophrenia.

Schizophrenia constitutes a complex disease. Negative and cognitive symptoms are enduring and debilitating components of the disorder, highly associated to disability and burden. Disrupted neurotransmission circuits in dorsolateral prefrontal cortex (DLPFC) have been related to these symptoms. To identify candidates altered in schizophrenia, we performed a pilot proteomic analysis on postmortem human DLPFC tissue from patients with schizophrenia (n=4) and control (n=4) subjects in a pool design using differential isotope peptide labelling followed by liquid chromatography tandem mass spectrometry (LC-MS/MS). We quantified 1315 proteins with two or more unique peptides, 116 of which showed altered changes. Of these altered proteins, we selected four with potential roles on cell signaling, neuronal development and synapse functioning for further validation: casein kinase I isoform epsilon (CSNK1E), fatty acid-binding protein 4 (FABP4), neurofilament triplet H protein (NEFH), and retinal dehydrogenase 1 (ALDH1A1). Immunoblot validation confirmed our proteomic findings of these proteins being decreased in abundance in the schizophrenia samples. Additionally, we conducted immunoblot validation of these candidates on an independent sample cohort comprising 23 patients with chronic schizophrenia and 23 matched controls. In this second cohort, CSNK1E, FABP4 and NEFH were reduced in the schizophrenia group while ALDH1A1 did not significantly change. This study provides evidence indicating these proteins are decreased in schizophrenia: CSNK1E, involved in circadian molecular clock signaling, FABP4 with possible implication in synapse functioning, and NEFH, important for cytoarchitecture organization. Hence, these findings suggest the possible implication of these proteins in the cognitive and/or negative symptoms in schizophrenia.

Specificity proteins 1 and 4, hippocampal volume and first-episode psychosis.

We assessed specificity protein 1 (SP1) and 4 (SP4) transcription factor levels in peripheral blood mononuclear cells and conducted a voxel-based morphometry analysis on brain structural magnetic resonance images from 11 patients with first-episode psychosis and 14 healthy controls. We found lower SP1 and SP4 levels in patients, which correlated positively with right hippocampal volume. These results extend previous evidence showing that such transcription factors may constitute a molecular pathway to the development of psychosis.

In utero undernutrition in male mice programs liver lipid metabolism in the second-generation offspring involving altered Lxra DNA methylation.

Obesity and type 2 diabetes have a heritable component that is not attributable to genetic factors. Instead, epigenetic mechanisms may play a role. We have developed a mouse model of intrauterine growth restriction (IUGR) by in utero malnutrition. IUGR mice developed obesity and glucose intolerance with aging. Strikingly, offspring of IUGR male mice also developed glucose intolerance. Here, we show that in utero malnutrition of F1 males influenced the expression of lipogenic genes in livers of F2 mice, partly due to altered expression of Lxra. In turn, Lxra expression is attributed to altered DNA methylation of its 5' UTR region. We found the same epigenetic signature in the sperm of their progenitors, F1 males. Our data indicate that in utero malnutrition results in epigenetic modifications in germ cells (F1) that are subsequently transmitted and maintained in somatic cells of the F2, thereby influencing health and disease risk of the offspring.

Reduced expression of SP1 and SP4 transcription factors in peripheral blood mononuclear cells in first-episode psychosis.

Alterations of transcription factor specificity protein 4 (SP4) and 1 (SP1) have been linked to different neuropsychiatric diseases. Reduced SP4 and SP1 protein levels in the prefrontal cortex have been associated with bipolar disorder and schizophrenia, respectively, suggesting that both factors could be involved in the pathogenesis of disorders with psychotic features. The aim of this study was to investigate whether the reduction of SP1, SP4 and SP3 protein and mRNA expression in peripheral blood mononuclear cells in the early stages of psychosis may act as a potential biomarker of these disorders. A cross-sectional study of first-episode psychosis patients (n = 14) compared to gender- and age-matched healthy controls (n = 14) was designed. Patients were recruited through the adult mental health services of Parc Sanitari Sant Joan de Déu. Protein and gene expression levels of SP1, SP4 and SP3 were assessed in peripheral blood mononuclear cells of patients with first-episode psychosis and healthy control subjects. We report that protein levels of SP1 and SP4, but not SP3, are significantly reduced in patients compared to controls. In contrast, we did not observe any differences in expression levels for SP1, SP4 or SP3 genes between patient and control groups. In patients, SP4 protein levels were significantly associated with SP1 protein levels. No association was found, however, between protein and gene expression levels for each factor. Our study shows reduced SP1 and SP4 protein levels in first-episode psychosis in lymphocytes, suggesting that these transcription factors are potential peripheral biomarkers of psychotic spectrum disorders in the early stages.

Analysis of Sp transcription factors in the postmortem brain of chronic schizophrenia: a pilot study of relationship to negative symptoms.

Negative symptoms are the most resilient manifestations in schizophrenia. An imbalance in dopamine and glutamate pathways has been proposed for the emergence of these symptoms. SP1, SP3 and SP4 transcription factors regulate genes in these pathways, suggesting a possible involvement in negative symptoms. In this study, we characterized Sp factors in the brains of subjects with schizophrenia and explored a possible association with negative symptoms. We also included analysis of NR1, NR2A and DRD2 as Sp target genes. Postmortem cerebellum and prefrontal cortex from an antemortem clinically well-characterized and controlled collection of elderly subjects with chronic schizophrenia (n = 16) and control individuals (n = 14) were examined. We used the Positive and Negative Syndrome and the Clinical Global Impression Schizophrenia scales, quantitative PCR and immunoblot. SP1 protein and mRNA were reduced in the prefrontal cortex in schizophrenia whereas none of Sp factors were altered in the cerebellum. However, we found that SP1, SP3 and SP4 protein levels inversely correlated with negative symptoms in the cerebellum. Furthermore, NR2A and DRD2 mRNA levels correlated with negative symptoms in the cerebellum. In the prefrontal cortex, SP1 mRNA and NR1 and DRD2 inversely correlated with these symptoms while Sp protein levels did not. This pilot study not only reinforces the involvement of SP1 in schizophrenia, but also suggests that reduced levels or function of SP1, SP4 and SP3 may participate in negative symptoms, in part through the regulation of NMDA receptor subunits and/or Dopamine D2 receptor, providing novel information about the complex negative symptoms in this disorder.

The transcription factor SP4 is reduced in postmortem cerebellum of bipolar disorder subjects: control by depolarization and lithium.

OBJECTIVES: Regulation of gene expression is important for the development and function of the nervous system. However, the transcriptional programs altered in psychiatric diseases are not completely characterized. Human gene association studies and analysis of mutant mice suggest that the transcription factor specificity protein 4 (SP4) may be implicated in the pathophysiology of psychiatric diseases. We hypothesized that SP4 levels may be altered in the brain of bipolar disorder (BD) subjects and regulated by neuronal activity and drug treatment. METHODS: We analyzed messenger RNA (mRNA) and protein levels of SP4 and SP1 in the postmortem prefrontal cortex and cerebellum of BD subjects (n = 10) and controls (n = 10). We also examined regulation of SP4 mRNA and protein levels by neuronal activity and lithium in rat cerebellar granule neurons. RESULTS: We report a reduction of SP4 and SP1 proteins, but not mRNA levels, in the cerebellum of BD subjects. SP4 protein and mRNA levels were also reduced in the prefrontal cortex. Moreover, we found in rat cerebellar granule neurons that under non-depolarizing conditions SP4, but not SP1, was polyubiquitinated and degraded by the proteasome while lithium stabilized SP4 protein. CONCLUSIONS: Our study provides the first evidence of altered SP4 protein in the cerebellum and prefrontal cortex in BD subjects supporting a possible role of transcription factor SP4 in the pathogenesis of the disease. In addition, our finding that SP4 stability is regulated by depolarization and lithium provides a pathway through which neuronal activity and lithium could control gene expression suggesting that normalization of SP4 levels could contribute to treatment of affective disorders.