Javier Martínez-Botas

Hydroxymethylglutaryl-coenzyme A reductase inhibition stimulates caspase-1 activity and Th1-cytokine release in peripheral blood mononuclear cells

T cells are prominent components of both early and late atherosclerotic lesions and the role of Th1/Th2 cells subsets in the evolution and rupture of the plaque is currently under investigation. Statins, which are inhibitors of 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase, exert actions beyond that of simply lowering cholesterol levels, and some effects on immune function have been reported. We studied in vitro the effects of fluvastatin on Th1/Th2 cytokine release in relation to caspase-1 activation, in human peripheral-blood mononuclear cells (PBMC) stimulated or not with Mycobacterium tuberculosis. Fluvastatin treatment resulted in the activation of caspase-1 and in a small secretion of interleukin (IL)-1beta, IL-18, and IFNgamma (Th1). In the presence of bacteria, the release of these cytokines was highly increased by the statin in a synergistic way. By contrast, production of IL-12, IL-10 and IL-4 were unaffected by the statin. Not only did mevalonate abolish the effects of the statin but it also prevented the caspase-1 activation induced by the bacteria, suggesting the involvement of isoprenoids in the response to M. tuberculosis. It is proposed that inhibition of HMG-CoA reductase may be immunoprotective by enhancing the Th1 response, which has therapeutical potential not only in atherosclerosis but also in infectious diseases.

Cholesterol starvation decreases P34cdc2 kinase activity and arrests the cell cycle at G2

As a major component of mammalian cell plasma membranes, cholesterol is essential for cell growth. Accordingly, the restriction of cholesterol provision has been shown to result in cell proliferation inhibition. We explored the potential regulatory role of cholesterol on cell cycle progression. MOLT‐4 and HL‐60 cell lines were cultured in a cholesterol‐deficient medium and simultaneously exposed to SKF 104976, which is a specific inhibitor of lanosterol 14‐α demethylase. Through HPLC analyses with on‐line radioactivity detection, we found that SKF 104976 efficiently blocked the [14C]‐acetate incorporation into cholesterol, resulting in an accumulation of lanosterol and dihydrolanosterol, without affecting the synthesis of mevalonic acid. The inhibitor also produced a rapid and intense inhibition of cell proliferation (IC50 = 0.1 µM), as assessed by both [3H]‐thymidine incorporation into DNA and cell counting. Flow cytometry and morphological examination showed that treatment with SKF 104976 for 48 h or longer resulted in the accumulation of cells specifically at G2 phase, whereas both the G1 traversal and the transition through S were unaffected. The G2 arrest was accompanied by an increase in the hyperphosphorylated form of p34cdc2 and a reduction of its activity, as determined by assaying the H1 histone phosphorylating activity of p34cdc2 immunoprecipitates. The persistent deficiency of cholesterol induced apoptosis. However, supplementing the medium with cholesterol, either in the form of LDL or free cholesterol dissolved in ethanol, completely abolished these effects, whereas mevalonate was ineffective. Caffeine, which abrogates the G2 checkpoint by preventing p34cdc2 phosphorylation, reduced the accumulation in G2 when added to cultures containing cells on transit to G2, but was ineffective in cells arrested at G2 by sustained cholesterol starvation. Cells arrested in G2, however, were still viable and responded to cholesterol provision by activating p34cdc2 and resuming the cell cycle. We conclude that in both lymphoblastoid and promyelocytic cells, cholesterol availability governs the G2 traversal, probably by affecting p34cdc2 activity.—Martínez‐Botas, J., Suárez, Y., Ferruelo, A. J., Gómez‐Coronado, D., Lasunción, M.A. Cholesterol starvation decreases P34cdc2 kinase activity and arrests the cell cycle at G2. FASEB J. 13, 1359–1370 (1999)

Induction of the endoplasmic reticulum stress protein GADD153/CHOP by capsaicin in prostate PC-3 cells: A microarray study

The effect of capsaicin, main pungent ingredient of hot chilli peppers, in the gene expression profile of human prostate PC-3 cancer cells has been analyzed using a microarray approach. We identified 10 genes that were down-regulated and five genes that were induced upon capsaicin treatment. The data obtained from microarray analysis were then validated using quantitative real-time PCR assays and Western blot analysis. The most remarkable change was the up-regulation of GADD153/CHOP, an endoplasmic reticulum stress-regulated gene. Activation of GADD153/CHOP protein was corroborated by immunofluorescence and Western blot. We then tested the contribution of GADD153/CHOP to protection against capsaicin-induced cell death using RNA interference. Blockage of GADD153/CHOP expression by small interfering RNA, significantly reduced capsaicin-induced cell death in PC-3 cells. Taken together, these results suggested that capsaicin induces the antiproliferative effect through a mechanism facilitated by ER stress in prostate PC-3 cells.

Dose-dependent effects of lovastatin on cell cycle progression. Distinct requirement of cholesterol and non-sterol mevalonate derivatives

The mevalonate pathway is tightly linked to cell proliferation. The aim of the present study is to determine the relationship between the inhibition of this pathway by lovastatin and the cell cycle. HL-60 and MOLT-4 human cell lines were cultured in a cholesterol-free medium and treated with increasing concentrations of lovastatin, and their effects on cell proliferation and the cell cycle were analyzed. Lovastatin was much more efficient in inhibiting cholesterol biosynthesis than protein prenylation. As a result of this, lovastatin blocked cell proliferation at any concentration used, but its effects on cell cycle distribution varied. At relatively low lovastatin concentrations (less than 10 microM), cells accumulated preferentially in G(2) phase, an effect which was both prevented and reversed by low-density lipoprotein cholesterol. At higher concentrations (50 microM), the cell cycle was also arrested at G(1) phase. In cells treated with lovastatin, those arrested at G(1) progressed through S upon mevalonate provision, whereas cholesterol supply allowed cells arrested at G(2) to traverse M phase. These results demonstrate the distinct roles of mevalonate, or its non-sterol derivatives, and cholesterol in cell cycle progression, both being required for normal cell cycling.

Gene expression profiling of subcutaneous adipose tissue in morbid obesity using a focused microarray: Distinct expression of cell-cycle- and differentiation-related genes

BackgroundObesity results from an imbalance between food intake and energy expenditure, which leads to an excess of adipose tissue. The excess of adipose tissue and adipocyte dysfunction associated with obesity are linked to the abnormal regulation of adipogenesis. The objective of this study was to analyze the expression profile of cell-cycle- and lipid-metabolism-related genes of adipose tissue in morbid obesity.MethodsWe used a custom-made focused cDNA microarray to determine the adipose tissue mRNA expression profile. Gene expression of subcutaneous abdominal fat samples from 15 morbidly obese women was compared with subcutaneous fat samples from 10 nonobese control patients. The findings were validated in an independent population of 31 obese women and 9 obese men and in an animal model of obesity (Lepob/ob mice) by real-time RT-PCR.ResultsMicroarray analysis revealed that transcription factors that regulate the first stages of adipocyte differentiation, such as CCAAT/enhancer binding protein beta (C/EBPβ) and JUN, were upregulated in the adipose tissues of morbidly obese patients. The expression of peroxisome proliferator-activated receptor gamma (PPARγ), a transcription factor which controls lipid metabolism and the final steps of preadipocyte conversion into mature adipocytes, was downregulated. The expression of three cyclin-dependent kinase inhibitors that regulate clonal expansion and postmitotic growth arrest during adipocyte differentiation was also altered in obese subjects: p18 and p27 were downregulated, and p21 was upregulated. Angiopoietin-like 4 (ANGPTL4), which regulates angiogenesis, lipid and glucose metabolism and it is know to increase dramatically in the early stages of adipocyte differentiation, was upregulated. The expression of C/EBPβ, p18, p21, JUN, and ANGPTL4 presented similar alterations in subcutaneous adipose tissue of Lepob/ob mice.ConclusionsOur microarray gene profiling study revealed that the expression of genes involved in adipogenesis is profoundly altered in the subcutaneous adipose tissue of morbidly obese subjects. This expression pattern is consistent with an immature adipocyte phenotype that could reflect the expansion of the adipose tissue during obesity.

Early and prolonged intake of partially hydrogenated fat alters the expression of genes in rat adipose tissue

OBJECTIVE Our previous study indicated that partially hydrogenated fat (PHF) diets, rich in trans-isomers, alter plasma lipids and increase the lipogenesis rate on adipose tissue in rats at a young age. In the present study we investigated the effects of dietary PHF on the expression of genes associated with glucose and lipid metabolism in rat adipose tissue. METHODS Female Wistar rats were fed normolipidic diets containing PHF (rich in trans-fatty acids and poor in polyunsaturated fatty acids [PUFAs]), soy oil (rich in omega-6 PUFAs), and fish oil (rich in omega-3 PUFAs) during gestation and lactation; young male pups were fed the same diets from weaning until 120 d of life. The mRNA expression of peroxisome proliferator-activated receptor-gamma, tumor necrosis factor-alpha, resistin, adiponectin, and leptin were analyzed in retroperitoneal adipose tissue (RET) using real time polymerase chain reaction. RESULTS The PHF group showed the highest triacylglycerol, glucose, and insulin levels and the lowest plasma adiponectin level. The RET of PHF incorporated trans-fatty acids, whereas fish oil and soy oil groups had increased omega-3 and omega-6 PUFAs, respectively. In the RET the PHF group had the highest resistin and tumor necrosis factor-alpha levels and the lowest adiponectin and peroxisome proliferator-activated receptor-gamma gene expressions, whereas the fish oil group had the highest peroxisome proliferator-activated receptor-gamma and the lowest leptin gene expression. CONCLUSION Prolonged intake of PHF has a negative effect on the expression of genes in RET when compared with diets with omega-6 and omega-3 PUFAs. These changes may be an effect of the smaller proportions of PUFAs in this fat, instead of being only caused by trans-fatty acids.

Identification of novel peptide biomarkers to predict safety and efficacy of cow's milk oral immunotherapy by peptide microarray

Cows milk oral immunotherapy (CM‐OIT) is still an experimental treatment. The development of novel biomarkers to predict the safety and efficacy of CM‐OIT is crucial to translate this treatment to common clinical practice.

Mapping of the IgE and IgG4 Sequential Epitopes of Ovomucoid with a Peptide Microarray Immunoassay

Background: Ovomucoid (Gal d 1) has been demonstrated to be the most important allergen in IgE-mediated egg allergy. Peptide microarray analysis is a novel method that can provide useful information on the nature of specific allergens. Methods: A peptide microarray immunoassay was performed using a 15- and 20-amino acid (aa) library of overlapping peptides (3-offset) of the primary sequence of ovomucoid. Sera from 50 patients with IgE-mediated egg allergy and reactivity to ovomucoid, with more than 1 year of follow-up, and sera from 10 controls were tested. Peptides were considered major epitopes when the average weighted Z-score was greater than 3 and recognized by at least 20% of the patient’s sera. Specific IgE epitopes were established on the basis of the IgE/IgG4 Z-score ratio. Results: The IgE and IgG4 recognition pattern was similar in both sets of peptides, but the signal intensity was generally higher in the 20-aa set. Thirty-four percent of the patients did not recognize any IgE sequential peptide and 20% of the patients recognized more than 10 sequential peptides. We identified 3 major IgE B-cell epitopes in domains I and II of ovomucoid. IgE/IgG4 ratio analysis showed that peptides 1–2 (aa 4–20) and peptides 29–31 (aa 91–104) were specific IgE epitopes. Conclusion: By using peptide microarray immunoassay in egg-allergic patients, we established that 34% of the patients do not have any linear epitope recognized by IgE. Further studies are needed to determine the clinical relevance of this finding.

Contribution of IncFII and Broad-Host IncA/C and IncN Plasmids to the Local Expansion and Diversification of Phylogroup B2 Escherichia coli ST131 Clones Carrying blaCTX-M-15 and qnrS1 Genes

ABSTRACT The recent increase of CTX-M-15-producing Escherichia coli isolates in our institution was caused by diverse clonal backgrounds, including mainly B2 sequence type 131 (ST131) clones presenting variable virulence profiles but also A1 (ST617, ST410), B1, and D1 (ST405) clones. Besides IncFII-pC15-1a, we detected multidrug-resistant IncA/C2 and IncN plasmids carrying blaCTX-M-15 and/or qnrS1. Our study highlights the diversification of highly transmissible resistant and virulent clones and the recombinogenic potential of broad-host plasmids contributing to the expansion of genetic regions coding for multidrug resistance to other bacterial lineages.

Dietary lipids modulate the expression of miR-107, an miRNA that regulates the circadian system

SCOPE The increased prevalence of cardiovascular diseases (CVDs) has been hypothesized to be the result of an increased exposure to a host of atherogenic environmental factors, paramount among them being unhealthy dietary habits. Long-chain n-3 polyunsaturated fatty acids have been shown to have cardio protective effects, partially due to their ability to regulate gene expression. In this regard, increasing attention has been devoted to the role of miRNAs as regulators of multiple metabolic pathways whose deregulation has been associated with CVD risk. METHODS AND RESULTS In this work, we investigated whether miRNA expression was regulated by docosahexanoic acid, conjugated linoleic acid, and cholesterol in Caco-2 cells. The modulated miRNAs, miR-107 was differentially expressed by all treatments and this modulation was independent of its hosting gene, PANK1, possibly through its own promoter, which contains binding sites for metabolically relevant transcription factors. Among the putative target genes of miR-107, we found some genes with key roles in circadian rhythm. Specifically, we demonstrated that binding of miR-107 to the CLOCK gene results in the deregulation of the circadian rhythm of the cells. CONCLUSION Since chronodisruption has been linked to metabolic disorders such as type 2 diabetes, atherosclerosis, obesity, and CVD, our findings suggests that miR-107 could represent a new approach for pharmacological treatment of these diseases.