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Perilipins are abundant lipid droplet (LD) proteins present in all metazoans and also in Amoebozoa and fungi. Humans express five perilipins, which share a similar domain organization: an amino-terminal PAT domain and an 11-mer repeat region, which can fold into amphipathic helices that interact with LDs, followed by a structured carboxy-terminal domain. Variations of this organization that arose during vertebrate evolution allow for functional specialization between perilipins in relation to the metabolic needs of different tissues. We discuss how different features of perilipins influence their interaction with LDs and their cellular targeting. PLIN1 and PLIN5 play a direct role in lipolysis by regulating the recruitment of lipases to LDs and LD interaction with mitochondria. Other perilipins, particularly PLIN2, appear to protect LDs from lipolysis, but the molecular mechanism is not clear. PLIN4 stands out with its long repetitive region, whereas PLIN3 is most widely expressed and is used as a nascent LD marker. Finally, we discuss the genetic variability in perilipins in connection with metabolic disease, prominent for PLIN1 and PLIN4, underlying the importance of understanding the molecular function of perilipins., (© 2023 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Background: The role of macrophages in the development of rhabdomyolysis-induced acute kidney injury (RM-AKI) has been established, but an in-depth understanding of the changes in the immune landscape could help to improve targeted strategies. Whereas senescence is usually associated with chronic kidney processes, we also wished to explore whether senescence could also occur in AKI and whether senolytics could act on immune cells., Methods: Single-cell RNA sequencing was used in the murine glycerol-induced RM-AKI model to dissect the transcriptomic characteristics of CD45+ live cells sorted from kidneys 2 days after injury. Public datasets from murine AKI models were reanalysed to explore cellular senescence signature in tubular epithelial cells (TECs). A combination of senolytics (dasatinib and quercetin, DQ) was administered to mice exposed or not to RM-AKI., Results: Unsupervised clustering of nearly 17 000 single-cell transcriptomes identified seven known immune cell clusters. Sub-clustering of the mononuclear phagocyte cells revealed nine distinct cell sub-populations differently modified with RM. One macrophage cluster was particularly interesting since it behaved as a critical node in a trajectory connecting one major histocompatibility complex class IIhigh (MHCIIhigh) cluster only present in Control to two MHCIIlow clusters only present in RM-AKI. This critical cluster expressed a senescence gene signature, that was very different from that of the TECs. Senolytic DQ treatment blocked the switch from a F4/80highCD11blow to F4/80lowCD11bhigh phenotype, which correlated with prolonged nephroprotection in RM-AKI., Conclusions: Single-cell RNA sequencing unmasked novel transitional macrophage subpopulation associated with RM-AKI characterized by the activation of cellular senescence processes. This work provides a proof-of-concept that senolytics nephroprotective effects may rely, at least in part, on subtle immune modulation., (© The Author(s) 2023. Published by Oxford University Press on behalf of the ERA.)

Duchenne muscular dystrophy (DMD) is a severe form of muscular dystrophy caused by mutations in the dystrophin gene. We characterized which isoforms of dystrophin were expressed by human induced pluripotent stem cell (hiPSC)-derived cardiac fibroblasts obtained from control and DMD patients. Distinct dystrophin isoforms were observed; however, highest molecular weight isoform was absent in DMD patients carrying exon deletions or mutations in the dystrophin gene. The loss of the full-length dystrophin isoform in hiPSC-derived cardiac fibroblasts from DMD patients resulted in deficient formation of actin microfilaments and a metabolic switch from mitochondrial oxidation to glycolysis. The DMD hiPSC-derived cardiac fibroblasts exhibited a dysregulated mitochondria network and reduced mitochondrial respiration, with enhanced compensatory glycolysis to sustain cellular ATP production. This metabolic remodeling was associated with an exacerbated myofibroblast phenotype and increased fibroblast activation in response to pro fibrotic challenges. As cardiac fibrosis is a critical pathological feature of the DMD heart, the myofibroblast phenotype induced by the absence of dystrophin may contribute to deterioration in cardiac function. Our study highlights the relationship between cytoskeletal dynamics, metabolism of the cell and myofibroblast differentiation and provides a new mechanism by which inactivation of dystrophin in non-cardiomyocyte cells may increase the severity of cardiopathy., (© 2023. The Author(s).)

Senescence is a key event in the impairment of adipose tissue (AT) function with obesity and aging but the underlying molecular and cellular players remain to be fully defined, particularly with respect to the human AT progenitors. We have found distinct profiles of senescent progenitors based on AT location between stroma from visceral versus subcutaneous AT. In addition to flow cytometry, we characterized the location differences with transcriptomic and proteomic approaches, uncovering the genes and developmental pathways that are underlying replicative senescence. We identified key components to include INBHA as well as SFRP4 and GREM1, antagonists for the WNT and BMP pathways, in the senescence-associated secretory phenotype and NOTCH3 in the senescence-associated intrinsic phenotype. Notch activation in AT progenitors inhibits adipogenesis and promotes myofibrogenesis independently of TGFβ. In addition, we demonstrate that NOTCH3 is enriched in the premyofibroblast progenitor subset, which preferentially accumulates in the visceral AT of patients with an early obesity trajectory. Herein, we reveal that NOTCH3 plays a role in the balance of progenitor fate determination preferring myofibrogenesis at the expense of adipogenesis. Progenitor NOTCH3 may constitute a tool to monitor replicative senescence and to limit AT dysfunction in obesity and aging., (© 2023 The Authors. Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

Myocardial infraction (MI) is the principal risk factor for the onset of heart failure (HF). Investigations regarding the physiopathology of MI progression to HF have revealed the concerted engagement of other tissues, such as the autonomic nervous system and the medulla oblongata (MO), giving rise to systemic effects, important in the regulation of heart function. Cardiac sympathetic afferent denervation following application of resiniferatoxin (RTX) attenuates cardiac remodelling and restores cardiac function following MI. While the physiological responses are well documented in numerous species, the underlying molecular responses during the initiation and progression from MI to HF remains unclear. We obtained multi-tissue time course proteomics with a murine model of HF induced by MI in conjunction with RTX application. We isolated tissue sections from the left ventricle (LV), MO, cervical spinal cord and cervical vagal nerves at four time points over a 12-week study. Bioinformatic analyses consistently revealed a high statistical enrichment for metabolic pathways in all tissues and treatments, implicating a central role of mitochondria in the tissue-cellular response to both MI and RTX. In fact, the additional functional pathways found to be enriched in these tissues, involving the cytoskeleton, vesicles and signal transduction, could be downstream of responses initiated by mitochondria due to changes in neuronal pulse frequency after a shock such as MI or the modification of such frequency communication from the heart to the brain after RTX application. Development of future experiments, based on our proteomic results, should enable the dissection of more precise mechanisms whereby metabolic changes in neuronal and cardiac tissues can effectively ameliorate the negative physiological effects of MI via RTX application., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Dysregulations of lipid metabolism in the liver may trigger steatosis progression, leading to potentially severe clinical consequences such as nonalcoholic fatty liver diseases (NAFLDs). Molecular mechanisms underlying liver lipogenesis are very complex and fine-tuned by chromatin dynamics and multiple key transcription factors. Here, we demonstrate that the nuclear factor HMGB1 acts as a strong repressor of liver lipogenesis. Mice with liver-specific Hmgb1 deficiency display exacerbated liver steatosis, while Hmgb1 -overexpressing mice exhibited a protection from fatty liver progression when subjected to nutritional stress. Global transcriptome and functional analysis revealed that the deletion of Hmgb1 gene enhances LXRα and PPARγ activity. HMGB1 repression is not mediated through nucleosome landscape reorganization but rather via a preferential DNA occupation in a region carrying genes regulated by LXRα and PPARγ. Together, these findings suggest that hepatocellular HMGB1 protects from liver steatosis development. HMGB1 may constitute a new attractive option to therapeutically target the LXRα-PPARγ axis during NAFLD.

Energetic metabolism controls key steps of kidney development, homeostasis, and epithelial repair following acute kidney injury (AKI). Hepatocyte nuclear factor-1β (HNF-1β) is a master transcription factor that controls mitochondrial function in proximal tubule (PT) cells. Patients with HNF1B pathogenic variant display a wide range of kidney developmental abnormalities and progressive kidney fibrosis. Characterizing the metabolic changes in PT cells with HNF-1β deficiency may help to identify new targetable molecular hubs involved in HNF1B-related kidney phenotypes and AKI. Here, we combined 1 H-NMR-based metabolomic analysis in a murine PT cell line with CrispR/Cas9-induced Hnf1b invalidation (Hnf1b -/- ), clustering analysis, targeted metabolic assays, and datamining of published RNA-seq and ChIP-seq dataset to identify the role of HNF-1β in metabolism. Hnf1b -/- cells grown in normoxic conditions display intracellular ATP depletion, increased cytosolic lactate concentration, increased lipid droplet content, failure to use pyruvate for energetic purposes, increased levels of tricarboxylic acid (TCA) cycle intermediates and oxidized glutathione, and a reduction of TCA cycle byproducts, all features consistent with mitochondrial dysfunction and an irreversible switch toward glycolysis. Unsupervised clustering analysis showed that Hnf1b -/- cells mimic a hypoxic signature and that they cannot furthermore increase glycolysis-dependent energetic supply during hypoxic challenge. Metabolome analysis also showed alteration of phospholipid biosynthesis in Hnf1b -/- cells leading to the identification of Chka, the gene coding for choline kinase α, as a new putative target of HNF-1β. HNF-1β shapes the energetic metabolism of PT cells and HNF1B deficiency in patients could lead to a hypoxia-like metabolic state precluding further adaptation to ATP depletion following AKI., (© 2021 Federation of American Societies for Experimental Biology.)

Aim: Impairments in cerebral structure and cognitive performance in chronic heart failure (CHF) are critical components of its comorbidity spectrum. Autonomic afferents that arise from cardiac sensory fibres show enhanced activity with CHF. Desensitization of these fibres by local application of resiniferatoxin (RTX) during myocardial infarction (MI) is known to prevent cardiac hypertrophy, sympathetic hyperactivity and CHF. Whether these afferents mediate cerebral allostasis is unknown., Methods: CHF was induced by myocardial infarction. To evaluate if cardiac afferents contribute to cerebral allostasis, RTX was acutely applied to the pericardial space in controls (RTX) and in MI treated animals (MI/RTX). Subjects were then evaluated in a series of behavioural tests recapitulating different symptoms of depressive disorders. Proteomics of the frontal cortices (FC) was performed to identify contributing proteins and pathways responsible for behavioural allostasis., Results: Desensitization of cardiac afferents relieves hallmarks of an anxio/depressive-like state in mice. Unique protein signatures and regulatory pathways in FCs isolated from each treatment reveal the degree of complexity inherent in the FC response to stresses originating in the heart. While cortices from the combined treatment (MI/RTX) did not retain protein signatures from the individual treatment groups, all three groups suffer dysregulation in circadian entrainment., Conclusion: CHF is comorbid with an anxio/depressive-like state and ablation of cardiac afferents relieves the despair phenotype. The strikingly different proteomic profiles observed in FCs suggest that MI and RTX lead to unique brain-signalling patterns and that the combined treatment, potentially through destructive interference mechanisms, most closely resembles controls., (© 2020 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.)

The incidence of disorders associated with low inflammatory state, such as chronic kidney disease, increases in the elderly. The accumulation of senescent cells during aging and the senescence-associated secretory phenotype, which leads to inflammaging, is known to be deleterious and account for progressive organ dysfunction. To date, the cellular actors implicated in chronic inflammation in the kidney during aging are still not well characterized. Using the DECyt method, based on hierarchical clustering of flow cytometry data, we showed that aging was associated with significant changes in stromal cell diversity in the kidney. In particular, we identified two cell populations up-regulated with aging, the mesenchymal stromal cell subset (kMSC) expressing CD73 and the monocyte-derived Ly6C +  CCR2 +  macrophage subset expressing pro-inflammatory cytokines. Aged CD73 +  kMSCs depicted senescence associated features with low proliferation rate, increased DNA damage foci and Ccl2 expression. Using co-cultures experiments, we showed that aged CD73 +  kMSC promoted monocyte activation and secretion of inflammatory cytokines albeit less efficiently than young CD73 +  kMSCs. In the context of ageing, increased frequency of CD73 + kMSC subpopulations could provide additional niche factors to newly recruited monocytes favoring a positive regulatory loop in response to local inflammation. Interfering with such partnership during aging could be a valuable approach to regulate kidney inflammaging and to limit the risk of developing chronic kidney disease in the elderly.

Under caloric restriction, bone marrow adipocytes (BM-Ads) do not decrease in size compared to white adipocytes, suggesting they harbor unique metabolic properties. We compare human primary BM-Ads with paired subcutaneous adipocytes (SC-Ads) using proteomic and lipidomic approaches. We find that, although SC-Ads and BM-Ads share similar morphological features, they possess distinct lipid metabolism. Although BM-Ad shows enrichment in proteins involved in cholesterol metabolism, correlating with increased free cholesterol content, proteins involved in lipolysis were downregulated. In particular, monoacylglycerol lipase expression is strongly reduced in BM-Ads, leading to monoacylglycerol accumulation. Consequently, basal and induced lipolytic responses are absent in BM-Ads, affirming their differences in metabolic fitness upon caloric restriction. These specific metabolic features are not recapitulated in vitro using common protocols to differentiate bone marrow mesenchymal stem cells. Thus, contrary to classical SC-Ads, BM-Ads display a specific lipid metabolism, as they are devoid of lipolytic activity and exhibit a cholesterol-orientated metabolism., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Aging is a major risk factor in the development of chronic diseases, especially cardiovascular diseases. Age-related organ dysfunction is strongly associated with the accumulation of senescent cells. Cardiac mesenchymal stromal cells (cMSCs), deemed part of the microenvironment, modulate cardiac homeostasis through their vascular differentiation potential and paracrine activity. Transcriptomic analysis of cMSCs identified age-dependent biological pathways regulating immune responses and angiogenesis. Aged cMSCs displayed a senescence program characterized by Cdkn2a expression, decreased proliferation and clonogenicity, and acquisition of a senescence-associated secretory phenotype (SASP). Increased CCR2-dependent monocyte recruitment by aged cMSCs was associated with increased IL-1ß production by inflammatory macrophages in the aging heart. In turn, IL-1ß induced senescence in cMSCs and mimicked age-related phenotypic changes such as decreased CD90 expression. The CD90+ and CD90- cMSC subsets had biased vascular differentiation potentials, and CD90+ cMSCs were more prone to acquire markers of the endothelial lineage with aging. These features were related to the emergence of a new cMSC subset in the aging heart, expressing CD31 and endothelial genes. These results demonstrate that cMSC senescence and SASP production are supported by the installation of an inflammatory amplification loop, which could sustain cMSC senescence and interfere with their vascular differentiation potentials., (© 2019 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

Double-strand breaks (DSBs) are extremely detrimental DNA lesions that can lead to cancer-driving mutations and translocations. Non-homologous end joining (NHEJ) and homologous recombination (HR) represent the two main repair pathways operating in the context of chromatin to ensure genome stability. Despite extensive efforts, our knowledge of DSB-induced chromatin still remains fragmented. Here, we describe the distribution of 20 chromatin features at multiple DSBs spread throughout the human genome using ChIP-seq. We provide the most comprehensive picture of the chromatin landscape set up at DSBs and identify NHEJ- and HR-specific chromatin events. This study revealed the existence of a DSB-induced monoubiquitination-to-acetylation switch on histone H2B lysine 120, likely mediated by the SAGA complex, as well as higher-order signaling at HR-repaired DSBs whereby histone H1 is evicted while ubiquitin and 53BP1 accumulate over the entire γH2AX domains., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Metabolic pathways, once seen as a mere consequence of cell states, have emerged as active players in dictating different cellular events such as proliferation, self-renewal, and differentiation. Several studies have reported a role for folate-dependent one-carbon (1C) metabolism in stem cells; however, its exact mode of action and how it interacts with other cues are largely unknown. Here, we report a link between the Eph:ephrin cell-cell communication pathway and 1C metabolism in controlling neural stem cell differentiation. Transcriptional and functional analyses following ephrin stimulation revealed alterations in folate metabolism-related genes and enzymatic activity. In vitro and in vivo data indicate that Eph-B forward signaling alters the methylation state of H3K4 by regulating 1C metabolism and locks neural stem cell in a differentiation-ready state. Our study highlights a functional link between cell-cell communication, metabolism, and epigenomic remodeling in the control of stem cell self-renewal., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

One of the major difficulties that arises when selecting aptamers containing a G-quadruplex is the correct amplification of the ssDNA sequence. Can aptamers containing a G-quadruplex be selected from a degenerate library using non-equilibrium capillary electrophoresis (CE) of equilibrium mixtures (NECEEM) along with high-throughput Illumina sequencing? In this article, we present some mismatches of the G-quadruplex T29 aptamer specific to thrombin, which was PCR amplified and sequenced by Illumina sequencing. Then, we show the proportionality between the number of sequenced molecules of T29 added to the library and the number of sequences obtained in Illumina sequencing, and we find that T29 sequences from this aptamer can be detected in a random library of ssDNA after the sample is fractionated by NECEEM, amplified by PCR, and sequenced. Treatment of the data by the counting of double-stranded DNA T29 sequences containing a maximum of two mismatches reveals a good correlation with the enrichment factor (f E ). This factor is the ratio of the number of aptamer sequences found in the collected complex sample divided by the total number of sequencing reads (aptamer and non-aptamer) plus the quantity of T29 molecules (spiked into a DNA library) injected into CE.

The ability of DNA double-strand breaks (DSBs) to cluster in mammalian cells has been a subject of intense debate in recent years. Here we used a high-throughput chromosome conformation capture assay (capture Hi-C) to investigate clustering of DSBs induced at defined loci in the human genome. The results unambiguously demonstrated that DSBs cluster, but only when they are induced within transcriptionally active genes. Clustering of damaged genes occurs primarily during the G1 cell-cycle phase and coincides with delayed repair. Moreover, DSB clustering depends on the MRN complex as well as the Formin 2 (FMN2) nuclear actin organizer and the linker of nuclear and cytoplasmic skeleton (LINC) complex, thus suggesting that active mechanisms promote clustering. This work reveals that, when damaged, active genes, compared with the rest of the genome, exhibit a distinctive behavior, remaining largely unrepaired and clustered in G1, and being repaired via homologous recombination in postreplicative cells.

Gut microbiota dysbiosis has been implicated in a variety of systemic disorders, notably metabolic diseases including obesity and impaired liver function, but the underlying mechanisms are uncertain. To investigate this question, we transferred caecal microbiota from either obese or lean mice to antibiotic-free, conventional wild-type mice. We found that transferring obese-mouse gut microbiota to mice on normal chow (NC) acutely reduces markers of hepatic gluconeogenesis with decreased hepatic PEPCK activity, compared to non-inoculated mice, a phenotypic trait blunted in conventional NOD2 KO mice. Furthermore, transferring of obese-mouse microbiota changes both the gut microbiota and the microbiome of recipient mice. We also found that transferring obese gut microbiota to NC-fed mice then fed with a high-fat diet (HFD) acutely impacts hepatic metabolism and prevents HFD-increased hepatic gluconeogenesis compared to non-inoculated mice. Moreover, the recipient mice exhibit reduced hepatic PEPCK and G6Pase activity, fed glycaemia and adiposity. Conversely, transfer of lean-mouse microbiota does not affect markers of hepatic gluconeogenesis. Our findings provide a new perspective on gut microbiota dysbiosis, potentially useful to better understand the aetiology of metabolic diseases., (© 2017 The Authors. Published under the terms of the CC BY 4.0 license.)

Periodontitis and type 2 diabetes are connected pandemic diseases, and both are risk factors for cardiovascular complications. Nevertheless, the molecular factors relating these two chronic pathologies are poorly understood. We have shown that, in response to a long-term fat-enriched diet, mice present particular gut microbiota profiles related to three metabolic phenotypes: diabetic-resistant (DR), intermediate (Inter), and diabetic-sensitive (DS). Moreover, many studies suggest that a dysbiosis of periodontal microbiota could be associated with the incidence of metabolic and cardiac diseases. We investigated whether periodontitis together with the periodontal microbiota may also be associated with these different cardiometabolic phenotypes. We report that the severity of glucose intolerance is related to the severity of periodontitis and cardiac disorders. In detail, alveolar bone loss was more accentuated in DS than Inter, DR, and normal chow-fed mice. Molecular markers of periodontal inflammation, such as TNF-α and plasminogen activator inhibitor-1 mRNA levels, correlated positively with both alveolar bone loss and glycemic index. Furthermore, the periodontal microbiota of DR mice was dominated by the Streptococcaceae family of the phylum Firmicutes, whereas the periodontal microbiota of DS mice was characterized by increased Porphyromonadaceae and Prevotellaceae families. Moreover, in DS mice the periodontal microbiota was indicated by an abundance of the genera Prevotella and Tannerella, which are major periodontal pathogens. PICRUSt analysis of the periodontal microbiome highlighted that prenyltransferase pathways follow the cardiometabolic adaptation to a high-fat diet. Finally, DS mice displayed a worse cardiac phenotype, percentage of fractional shortening, heart rhythm, and left ventricle weight-to-tibia length ratio than Inter and DR mice. Together, our data show that periodontitis combined with particular periodontal microbiota and microbiome is associated with metabolic adaptation to a high-fat diet related to the severity of cardiometabolic alteration., (Copyright © 2016 the American Physiological Society.)

The expression and role of RNA binding proteins (RBPs) controlling mRNA translation during tumor progression remains largely uncharacterized. Analysis by immunohistochemistry of the expression of hnRNP A1, hnRNPH, RBM9/FOX2, SRSF1/ASF/SF2, SRSF2/SC35, SRSF3/SRp20, SRSF7/9G8 in breast tumors shows that the expression of hnRNP A1, but not the other tested RBPs, is associated with metastatic relapse. Strikingly, hnRNP A1, a nuclear splicing regulator, is also present in the cytoplasm of tumor cells of a subset of patients displaying exceedingly worse prognosis. Expression of a cytoplasmic mutant of hnRNP A1 leads to increased translation of the mRNA encoding the tyrosine kinase receptor RON/MTS1R, known for its function in tumor dissemination, and increases cell migration in vitro. hnRNP A1 directly binds to the 5' untranslated region of the RON mRNA and activates its translation through G-quadruplex RNA secondary structures. The correlation between hnRNP A1 and RON tumoral expression suggests that these findings hold clinical relevance.

DNA double-strand breaks (DSBs) elicit the so-called DNA damage response (DDR), largely relying on ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PKcs), two members of the PI3K-like kinase family, whose respective functions during the sequential steps of the DDR remains controversial. Using the DIvA system (DSB inducible via AsiSI) combined with high-resolution mapping and advanced microscopy, we uncovered that both ATM and DNA-PKcs spread in cis on a confined region surrounding DSBs, independently of the pathway used for repair. However, once recruited, these kinases exhibit non-overlapping functions on end joining and γH2AX domain establishment. More specifically, we found that ATM is required to ensure the association of multiple DSBs within "repair foci." Our results suggest that ATM acts not only on chromatin marks but also on higher-order chromatin organization to ensure repair accuracy and survival., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

A high-fat diet (HFD) induces metabolic disease and low-grade metabolic inflammation in response to changes in the intestinal microbiota through as-yet-unknown mechanisms. Here, we show that a HFD-derived ileum microbiota is responsible for a decrease in Th17 cells of the lamina propria in axenic colonized mice. The HFD also changed the expression profiles of intestinal antigen-presenting cells and their ability to generate Th17 cells in vitro. Consistent with these data, the metabolic phenotype was mimicked in RORγt-deficient mice, which lack IL17 and IL22 function, and in the adoptive transfer experiment of T cells from RORγt-deficient mice into Rag1-deficient mice. We conclude that the microbiota of the ileum regulates Th17 cell homeostasis in the small intestine and determines the outcome of metabolic disease., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Rhabdomyolysis can be life threatening if complicated by AKI. Macrophage infiltration has been observed in rat kidneys after glycerol-induced rhabdomyolysis, but the role of macrophages in rhabdomyolysis-induced AKI remains unknown. Here, in a patient diagnosed with rhabdomyolysis, we detected substantial macrophage infiltration in the kidney. In a mouse model of rhabdomyolysis-induced AKI, diverse renal macrophage phenotypes were observed depending on the stage of the disease. Two days after rhabdomyolysis, F4/80(low)CD11b(high)Ly6b(high)CD206(low) kidney macrophages were dominant, whereas by day 8, F4/80(high)CD11b(+)Ly6b(low)CD206(high) cells became the most abundant. Single-cell gene expression analyses of FACS-sorted macrophages revealed that these subpopulations were heterogeneous and that individual cells simultaneously expressed both M1 and M2 markers. Liposomal clodronate-mediated macrophage depletion significantly reduced the early infiltration of F4/80(low)CD11b(high)Ly6b(high)CD206(low) macrophages. Furthermore, transcriptionally regulated targets potentially involved in disease progression, including fibronectin, collagen III, and chemoattractants that were identified via single-cell analysis, were verified as macrophage-dependent in situ. In vitro, myoglobin treatment induced proximal tubular cells to secrete chemoattractants and macrophages to express proinflammatory markers. At day 30, liposomal clodronate-mediated macrophage depletion reduced fibrosis and improved both kidney repair and mouse survival. Seven months after rhabdomyolysis, histologic lesions were still present but were substantially reduced with prior depletion of macrophages. These results suggest an important role for macrophages in rhabdomyolysis-induced AKI progression and advocate the utility of long-term follow-up for patients with this disease., (Copyright © 2015 by the American Society of Nephrology.)

Obesity-associated inflammation contributes to the development of metabolic diseases. Although brite adipocytes have been shown to ameliorate metabolic parameters in rodents, their origin and differentiation remain to be characterized in humans. Native CD45-/CD34+/CD31- cells have been previously described as human adipocyte progenitors. Using two additional cell surface markers, MSCA1 (tissue nonspecific alkaline phosphatase) and CD271 (nerve growth factor receptor), we are able to partition the CD45-/CD34+/CD31- cell population into three subsets. We establish serum-free culture conditions without cell expansion to promote either white/brite adipogenesis using rosiglitazone, or bone morphogenetic protein 7 (BMP7), or specifically brite adipogenesis using 3-isobuthyl-1-methylxanthine. We demonstrate that adipogenesis leads to an increase of MSCA1 activity, expression of white/brite adipocyte-related genes, and mitochondriogenesis. Using pharmacological inhibition and gene silencing approaches, we show that MSCA1 activity is required for triglyceride accumulation and for the expression of white/brite-related genes in human cells. Moreover, native immunoselected MSCA1+ cells exhibit brite precursor characteristics and the highest adipogenic potential of the three progenitor subsets. Finally, we provided evidence that MSCA1+ white/brite precursors accumulate with obesity in subcutaneous adipose tissue (sAT), and that local BMP7 and inflammation regulate brite adipogenesis by modulating MSCA1 in human sAT. The accumulation of MSCA1+ white/brite precursors in sAT with obesity may reveal a blockade of their differentiation by immune cells, suggesting that local inflammation contributes to metabolic disorders through impairment of white/brite adipogenesis. Stem Cells 2015;33:1277-1291., (© 2014 AlphaMed Press.)

Weight control diets favorably affect parameters of the metabolic syndrome and delay the onset of diabetic complications. The adaptations occurring in adipose tissue (AT) are likely to have a profound impact on the whole body response as AT is a key target of dietary intervention. Identification of environmental and individual factors controlling AT adaptation is therefore essential. Here, expression of 271 transcripts, selected for regulation according to obesity and weight changes, was determined in 515 individuals before, after 8-week low-calorie diet-induced weight loss, and after 26-week ad libitum weight maintenance diets. For 175 genes, opposite regulation was observed during calorie restriction and weight maintenance phases, independently of variations in body weight. Metabolism and immunity genes showed inverse profiles. During the dietary intervention, network-based analyses revealed strong interconnection between expression of genes involved in de novo lipogenesis and components of the metabolic syndrome. Sex had a marked influence on AT expression of 88 transcripts, which persisted during the entire dietary intervention and after control for fat mass. In women, the influence of body mass index on expression of a subset of genes persisted during the dietary intervention. Twenty-two genes revealed a metabolic syndrome signature common to men and women. Genetic control of AT gene expression by cis signals was observed for 46 genes. Dietary intervention, sex, and cis genetic variants independently controlled AT gene expression. These analyses help understanding the relative importance of environmental and individual factors that control the expression of human AT genes and therefore may foster strategies aimed at improving AT function in metabolic diseases., Competing Interests: The authors have declared that no competing interests exist.

Genomic DNA (gDNA) contamination is an inherent problem during RNA purification that can lead to non-specific amplification and aberrant results in reverse transcription quantitative PCR (RT-qPCR). Currently, there is no alternative to RT(-) controls to evaluate the impact of the gDNA background on RT-PCR data. We propose a novel method (ValidPrime) that is more accurate than traditional RT(-) controls to test qPCR assays with respect to their sensitivity toward gDNA. ValidPrime measures the gDNA contribution using an optimized gDNA-specific ValidPrime assay (VPA) and gDNA reference sample(s). The VPA, targeting a non-transcribed locus, is used to measure the gDNA contents in RT(+) samples and the gDNA reference is used to normalize for GOI-specific differences in gDNA sensitivity. We demonstrate that the RNA-derived component of the signal can be accurately estimated and deduced from the total signal. ValidPrime corrects with high precision for both exogenous (spiked) and endogenous gDNA, contributing ∼60% of the total signal, whereas substantially reducing the number of required qPCR control reactions. In conclusion, ValidPrime offers a cost-efficient alternative to RT(-) controls and accurately corrects for signals derived from gDNA in RT-qPCR.

Glucocorticoids are frequently prescribed drugs with important side-effects such as glucose intolerance and tissue remodeling. The goal was to explore the molecular basis of the response of skeletal muscle and adipose tissue during a short-term dexamethasone treatment to better understand the induction of side-effects of glucocorticoids on these metabolic tissues. Fifteen healthy male subjects were assigned to a 4-day treatment with dexamethasone at 4 mg/day. The primary outcome measures were changes in gene expression profiling of subcutaneous skeletal muscle and adipose tissue. Urinary cortisol, plasma, and metabolic biochemistry were also assessed. In both tissues the prominent observation was a response to stress and increased inflammatory responses. An upregulation of the serum amyloid A was detected in skeletal muscle, adipose tissue, and plasma, whereas circulating levels of C reactive protein, another acute phase protein, decreased along with a worsened insulin sensitivity index. As tissue-specific features, tissue remodeling was shown in skeletal muscle while the adipose tissue exhibited a decreased energy metabolism. Several limitations might be raised due to the small number of subjects investigated: a possible cross talk with the mineralocorticoid receptor, and a single time point may not identify regulations occurring during longitudinal treatment. In line with the known physiological effect of glucocorticoids the early modulation of stress response genes was observed. An unexpected feature was the upregulation of the inflammatory and immune pathways. The identification of novel impact on two glucocorticoid target tissues provides a molecular basis for the design of more specific glucocorticoids devoid of adverse effects.

Chromatin undergoes major remodeling around DNA double-strand breaks (DSB) to promote repair and DNA damage response (DDR) activation. We recently reported a high-resolution map of γH2AX around multiple breaks on the human genome, using a new cell-based DSB inducible system. In an attempt to further characterize the chromatin landscape induced around DSBs, we now report the profile of SMC3, a subunit of the cohesin complex, previously characterized as required for repair by homologous recombination. We found that recruitment of cohesin is moderate and restricted to the immediate vicinity of DSBs in human cells. In addition, we show that cohesin controls γH2AX distribution within domains. Indeed, as we reported previously for transcription, cohesin binding antagonizes γH2AX spreading. Remarkably, depletion of cohesin leads to an increase of γH2AX at cohesin-bound genes, associated with a decrease in their expression level after DSB induction. We propose that, in agreement with their function in chromosome architecture, cohesin could also help to isolate active genes from some chromatin remodelling and modifications such as the ones that occur when a DSB is detected on the genome., Competing Interests: The authors have declared that no competing interests exist.

Of critical importance in the stress response is the post-transcriptional control of the expression of important genes involved in the control of cell survival and apoptosis. Here we report that miR-19, an oncogenic component of the miR-17-92/Oncomir-1 microRNA polycistron, regulates the expression of Ras homolog B (RhoB) in keratinocytes upon exposure to ultraviolet (UV) radiation. Strikingly, we could not find any evidence for deregulated expression of miR-19 during UV treatment. However, we show that miR-19-mediated regulation of antiapoptotic RhoB expression requires the binding of human antigen R (HuR), an AU-rich element binding protein, to the 3'-untranslated region of the rhoB mRNA. We propose that the loss of the interdependent binding between HuR and miR-19 to the rhoB mRNA upon UV exposure relieves this mRNA from miR-19-dependent inhibition of translation and contributes to the apoptotic response.

DNA double strand breaks (DSBs) are among the most deleterious forms of lesions and deciphering the details of the chromatin landscape induced around DSBs represents a great challenge for molecular biologists. Chromatin Immunoprecipitation, followed by microarray hybridisation (ChIP-chip) or high-throughput sequencing (ChIP-seq), are powerful techniques that provide high-resolution maps of protein-genome interactions. However, applying these techniques to study chromatin changes induced around DSBs was previously hindered due to a lack of suitable DSB induction techniques. We have recently developed an experimental system utilizing a restriction enzyme fused to a modified oestrogen receptor ligand binding domain (AsiSI-ER), which generates multiple, sequence-specific and unambiguously positioned DSBs across the genome upon induction with 4-hydroxytamoxifen (4OHT).(1) Cell lines expressing this construct represent a powerful tool to study specific chromatin changes during DSB repair, enabling high-resolution profiling of DNA repair complexes and chromatin modifications induced around DSBs. Using this system, we have recently produced the first map of gammaH2AX, a DSB-induced chromatin modification, on two human chromosomes and have investigated its spreading properties.(1) Here we provide additional data characterizing the cell lines, present a genome-wide profile of gammaH2AX obtained by ChIP-seq, and discuss the potential of our system towards investigations of previously uncharacterized aspects of DSB repair.

Chromatin acts as a key regulator of DNA-related processes such as DNA damage repair. Although ChIP-chip is a powerful technique to provide high-resolution maps of protein-genome interactions, its use to study DNA double strand break (DSB) repair has been hindered by the limitations of the available damage induction methods. We have developed a human cell line that permits induction of multiple DSBs randomly distributed and unambiguously positioned within the genome. Using this system, we have generated the first genome-wide mapping of gammaH2AX around DSBs. We found that all DSBs trigger large gammaH2AX domains, which spread out from the DSB in a bidirectional, discontinuous and not necessarily symmetrical manner. The distribution of gammaH2AX within domains is influenced by gene transcription, as parallel mappings of RNA Polymerase II and strand-specific expression showed that gammaH2AX does not propagate on active genes. In addition, we showed that transcription is accurately maintained within gammaH2AX domains, indicating that mechanisms may exist to protect gene transcription from gammaH2AX spreading and from the chromatin rearrangements induced by DSBs.

Aims/hypothesis: Uncoupling protein (UCP) 3 is an inner mitochondrial membrane transporter mainly produced in skeletal muscle in humans. UCP3 plays a role in fatty acid metabolism and energy homeostasis and modulates insulin sensitivity. In humans, UCP3 content is higher in fast-twitch glycolytic muscle than in slow-twitch oxidative muscle and is dysregulated in type 2 diabetes. Here, we studied the molecular mechanisms determining human UCP3 levels in skeletal muscle and their regulation by fasting in transgenic mice., Methods: We produced a series of transgenic lines with constructs bearing different putative regulatory regions of the human UCP3 gene, including promoter and intron sequences. UCP3 mRNA and reporter gene expression and activity were measured in different skeletal muscles and tissues., Results: The profile of expression and the response to fasting and thyroid hormone of human UCP3 mRNA in transgenic mice with 16 kb of the human UCP3 gene were similar to that of the endogenous human gene. Various parts of the UCP3 promoter did not confer expression in transgenic lines. Inclusion of intron 1 resulted in an expression profile in skeletal muscle that was identical to that of human UCP3 mRNA. Further dissection of intron 1 revealed that distinct regions were involved in skeletal muscle expression, distribution among fibre types and response to fasting., Conclusions/interpretation: The control of human UCP3 transcription in skeletal muscle is not solely conferred by the promoter, but depends on several cis-acting elements in intron 1, suggesting a complex interplay between the promoter and intronic sequences.

Experiments with EMCV (Encephalomyocarditis virus) internal ribosome entry sites (IRESes) have shown that microRNAs (miRs) are unable to inhibit IRES driven translation. However, it is accepted that miRs can inhibit translation through multiple mechanisms, only some of which require interaction with the 5' cap structure. In this report, we first validate the targeting of miR-16 to a predicted binding site in the VEGF 3'UTR. We developed a series of experiments to ascertain whether or not miR-16 can inhibit translation of transcripts driven by either of the VEGF IRESes. Our results indicate that cellular IRESes can be classified as both sensitive and insensitive to miR control. While VEGF IRES-A activity was not altered by miR-16 targeting to the 3'UTR, IRES-B was susceptible to miR-16 inhibition. Taken together with previous results that show that IRES-B selectively translates the CUG initiated VEGF-121 isoform, we can conclude that the existence of two differentially susceptible IRESes in the VEGF 5'UTR leads to even more complex regulatory control of VEGF isoform production. This study demonstrates for the first time the inhibition of cellular IRES driven translation by a miR.

Vascular endothelial growth factor-A (VEGF), a powerful factor involved in vasculogenesis and angiogenesis, is translationally regulated through 2 independent internal ribosome entry sites (IRESs A and B). IRESs enable an mRNA to be translated under conditions in which 5'-cap-dependent translation is inhibited, such as low oxygen stress. In the VEGF mRNA, IRES A influences translation at the canonical AUG codon, whereas the 5' IRES B element regulates initiation at an upstream, in frame CUG. In this study, we have developed transgenic mice expressing reporter genes under the control of these 2 IRESs. We reveal that although these IRESs display low activity in embryos and adult tissues, they permit efficient translation at early time points in ischemic muscle, a stress under which cap-dependent translation is inhibited. These results demonstrate the in vivo efficacy of the VEGF IRESs in response to a local environmental stress such as hypoxia.

Targets of the oncogenic transcription factor v-Jun in the murine cell line C3H 10T1/2 cells have been identified using DNA microarrays. Two targets, Akap12 and Marcks, are downregulated in transformed cells and are known tumor suppressor genes. Overexpression of either Akap12 or Marcks in v-Jun-transformed cells reverses the transformed phenotype and leads to the re-expression of the other tumor suppressor gene, suggesting that these two genes cooperate in the establishment of the nontransformed state. Reverted cells continue to express v-Jun at high levels and also re-express c-Jun, which is normally repressed by v-Jun. A panel of six cell lines has been generated to evaluate the expression levels of other v-Jun targets in 10T1/2 cells. With these cells, we find that the upregulated target Sprr1a has an expression pattern that correlates with the transformed phenotype., (Copyright 2004 Nature Publishing Group)

The vascular endothelial growth factor-A (VEGF) gene locus contains eight exons that span 14 kb. Alternative splicing generates multiple, different mRNAs that in turn translate into at least five protein isoforms. While the canonical AUG start codon is located at position 1039 in exon 1, there also exists an upstream, in-frame CUG initiation codon that drives expression of L-VEGF, containing an additional 180 amino acids. Two separate internal ribosome entry sites (IRES) regulate the activity of each initiation codon. Thus the 5'-UTR of VEGF, which comprises the majority of exon 1, consists of IRES B, the CUG, IRES A, and the AUG, from 5' to 3'. Previously, it has been shown that IRES B regulates initiation at the CUG and IRES A regulates AUG usage. In this study, we have found evidence that the exon content of the VEGF mRNA, determined through alternative splicing, controls IRES A activity. While the CUG is most efficient at initiating translation, transcripts that lack both exons 6 and 7 and therefore contain an exon 5/8 junction lack AUG-initiated translation. The process of splicing is not responsible for this start codon selection since transfection of genomic and cDNA VEGF sequences give the same expression pattern. We hypothesize that long range tertiary interactions in the VEGF mRNA regulate IRES activity and thus control start codon selection. This is the first report describing the influence of alternatively spliced coding sequences on codon selection by modulating IRES activity.

BARX2 is a homeobox transcription factor that influences cellular differentiation in various developmental contexts. To begin to identify the gene targets that mediate its effects, chromatin immunoprecipitation (ChIP) was used to isolate BARX2 binding sites from the human MCF7 breast cancer cell line. Cloning and sequencing of BARX2-ChIP-derived DNA fragments identified 60 potential BARX2 target loci that were proximal to or within introns of genes involved in cytoskeletal organization, cell adhesion, growth factor signaling, transcriptional regulation, and RNA metabolism. The sequences of over half of the fragments showed homology with the mouse genome, and several sequences could be mapped to orthologous human and mouse genes. Binding of BARX2 to 21 genomic loci examined was confirmed quantitatively by replicate ChIP assays. A combination of sequence analysis and electrophoretic mobility shift assays revealed homeodomain binding sites within several fragments that bind to BARX2 in vitro. The majority of BARX2 binding fragments tested (14/19), also affected transcription in luciferase reporter gene assays. Mutation analyses of three fragments showed that their transcriptional activities required the HBS, and suggested that BARX2 regulates gene expression by binding to DNA elements containing paired TAAT motifs that are separated by a poly(T) sequence. Inhibition of BARX2 expression in MCF7 cells led to reduced expression of eight genes associated with BARX2 binding sites, indicating that BARX2 directly regulates their expression. The data suggest that BARX2 can coordinate the expression of a network of genes that influence the growth of MCF7 cells.

Studies of the fission yeast Schizosaccharomyces pombe have made major contributions towards understanding cell-cycle control and many other important aspects of cell biology. A series of pREP expression vectors that utilize the thiamine-repressible nmt1 promoter are used routinely to manipulate the expression of genes in fission yeast. A shortcoming of the nmt1 promoter is that it is very slowly induced following removal of thiamine from the growth medium, requiring approx. 16h for full induction. Invertase, an enzyme responsible for sucrose metabolism, is regulated transcriptionally by glucose derepression in S. pombe. Using the inv1 promoter, we have developed the pINV1 set of inducible protein expression vectors. A shift from glucose to sucrose-based culture medium leads to a very rapid induction of the inv1 promoter. Genes that are regulated by the inv1 promoter are fully induced within 1h of the shift to sucrose-based medium. The pINV1 vectors utilize a simple induction protocol and enable studies in fission yeast requiring tight and rapid regulation of protein synthesis.

DF-1 is a continuous cell line of chicken embryo fibroblasts. The cells are free of endogenous sequences related to avian sarcoma and leukosis viruses and have normal fibroblastic morphology. DF-1 cells support the replication of avian retroviruses; diverse oncogenes induce foci of oncogenic transformation on monolayers of DF-1, and avian leukosis viruses of envelope subgroups B, D, and C induce cell death and form plaques. The new cell line will greatly facilitate studies on oncogenic transformation and cell killing by avian viruses., (Copyright 1998 Academic Press.)

We have analysed differential gene expression in v-jun-transformed chicken embryo fibroblasts (CEF) compared to normal CEF by using the directional tag PCR subtraction method. From a first generation of putative Jun targets four clones were selected for study; they are upregulated in jun-transformed cells. Three of these clones showed homology to known genes: glutaredoxin, growth associated protein (GAP)-43/neuromodulin, and phenobarbital-induced cytochrome P450. The expression of these genes was analysed in fibroblasts transformed by various oncogenes. Expression of the glutaredoxin mRNA could be induced by a Jun-estrogen receptor chimaera in the absence of de novo protein biosynthesis. Based on this observation we conclude that glutaredoxin is a direct target of v-Jun.

The v-jun oncogene encodes a nuclear DNA binding protein that functions as a transcription factor and is part of the activator protein 1 complex. Oncogenic transformation by v-jun is thought to be mediated by the aberrant expression of specific target genes. To identify such Jun-regulated genes and to explore the mechanisms by which Jun affects their expression, we have fused the full-length v-Jun and an amino-terminally truncated form of v-Jun to the hormone-binding domain of the human estrogen receptor. The two chimeric proteins function as ligand-inducible transactivators. Expression of the fusion proteins in chicken embryo fibroblasts causes estrogen-dependent transformation.

The retroviral oncogene qin codes for a protein that belongs to the family of the winged helix transcription factors. The viral Qin protein, v-Qin, differs from its cellular counterpart, c-Qin, by functioning as a stronger transcriptional repressor and a more efficient inducer of tumors. This observation suggests that repression may be important in tumorigenesis. To test this possibility, chimeric proteins were constructed in which the Qin DNA-binding domain was fused to either a strong repressor domain (derived from the Drosophila Engrailed protein) or a strong activator domain (from the herpes simplex virus VP16 protein). The chimeric transcriptional repressor, Qin-Engrailed, transformed chicken embryo fibroblasts in culture and induced sarcomas in young chickens. The chimeric activator, Qin-VP16, failed to transform cells in vitro or in vivo and caused cellular resistance to oncogenic transformation by Qin. These data support the conclusion that the Qin protein induces oncogenic transformation by repressing the transcription of genes which function as negative growth regulators or tumor suppressors.

The chicken winged helix proteins, CWH-1, CWH-2 and CWH-3, were isolated and identified by homology cloning using the winged helix sequence of the retroviral oncogene qin as a probe. The CWH proteins act as growth stimulators in chicken embryo fibroblasts and in this activity resemble the Qin protein. Qin is a transcriptional regulator that functions as a repressor, and its oncogenic potential is correlated with the ability to repress transcription. In this communication we show that CWH proteins are localized in the cell nucleus, recognize the Qin DNA binding site and also function as transcriptional repressors. The repression activity of CWH-3 was mapped to the region of amino acids 211 to 311, a domain that is homologous to the major repression domain of Qin.

Winged helix transcription factors act as important regulators of embryonal development and tissue differentiation in vertebrates and invertebrates. Identification of the retroviral oncogene v-qin as a member of the winged helix family showed that these developmental regulators also have oncogenic potential. We used low-stringency hybridization of a chicken embryonic cDNA library to isolate cDNA clones coding for the three chicken winged helix (CWH) proteins, CWH-1, CWH-2, and CWH-3. The CWH genes are transcribed in a tissue-restricted pattern in adult and embryonic chicken tissues. The CWH proteins bind to conserved DNA binding sites for winged helix proteins in a sequence-specific manner. Expression of the CWH proteins from replication-competent retroviral RCAS vectors induces changes in morphology and growth pattern of chicken embryo fibroblasts. CWH-1 and CWH-3 also induce anchorage-independent growth in agar. Chicken embryo fibroblasts expressing the RCAS constructs release replication-competent viruses that are able to elicit the same cellular changes as the parental plasmid DNA. Our results suggest that winged helix transcription factors not only function as regulators of development and differentiation but also have the potential to stimulate abnormal cell proliferation.

The four nuclear factor I genes (NFI-A, NFI-B, NFI-C, and NFI-X) give rise to multiple isoforms by alternative splicing in many tissues. These NFI proteins cooperate with AP-1, Myc, and other transcription factors in regulating transcription of numerous cellular and viral genes. We have investigated the growth-regulatory potential of NFI by overexpressing cDNAs from chicken NFI genes -A, -B, -C, and -X in chicken embryo fibroblasts (CEF). None of the NFI cDNAs induced oncogenic transformation of CEF. However, overexpression of each of the NFI proteins caused similar morphological alteration of the cells, inducing them to become flattened and polygonal and to show increased adherence. The growth properties of these cells were similar to normal CEF. When these morphologically altered CEF were challenged by superinfection with oncogenic retroviruses, they were resistant to transformation by the nuclear oncogenes jun, fos, junD, myc, and qin but were readily transformed by cytoplasmic oncogenes src, mil/raf, ras, and fps. The NFI-A1 protein was able to alter transactivation by the cellular and viral Jun proteins in a promoter-dependent manner. The changes in cell morphology and reduced susceptibility to nuclear oncogenes were not seen with a carboxy-terminal truncation in the transactivation domain of NFI, suggesting that this region of the protein is essential for the observed effects. The dichotomy between the activities of nuclear and of cytoplasmic oncogenes in this system is discussed.