Stable conditional expression and effect of C/EBPβ-LIP in adipocytes using the pSLIK system

Murine 3T3-L1 adipocytes are widely used as a cellular model of obesity. However, whereas transfection of 3T3-L1 preadipocytes is straightforward, ectopic gene expression in mature 3T3-L1 adipocytes has proved challenging. Here, we used the pSLIK vector system to generate stable doxycycline-inducible expression of the liver-enriched inhibitor protein isoform of CCAAT/enhancer binding protein (C/EBP) {beta} (C/EBP{beta}-LIP) in fully differentiated 3T3-L1 adipocytes. Because overexpression of C/EBP{beta}-LIP impairs adipocyte differentiation, the C/EBP{beta}-LIP construct was first integrated in 3T3-L1 preadipocytes but expression was induced only when adipocytes were fully differentiated. Increased C/EBP{beta}-LIP in mature adipocytes down-regulated C/EBP{beta} target genes including 11{beta}-hydroxysteroid dehydrogenase type 1, phosphoenolpyruvate carboxykinase and fatty acid binding protein 4, but had no effect on asparagine synthetase, demonstrating that transcriptional down-regulation by C/EBP{beta}-LIP in 3T3-L1 adipocytes is not a general effect. Importantly, these genes were modulated in a similar manner in adipose tissue of mice with genetically increased C/EBP{beta}-LIP levels. The use of the pSLIK system to conditionally express transgenes in 3T3-L1 cells could be a valuable tool to dissect adipocyte physiology

USH2A is a Meissner’s corpuscle protein necessary for normal vibration sensing in mice and humans

Fingertip mechanoreceptors comprise sensory neuron endings together with specialized skin cells that form the end-organ. Exquisitely sensitive, vibration-sensing neurons are associated with Meissner’s corpuscles in the skin. In the present study, we found that USH2A, a transmembrane protein with a very large extracellular domain, was found in terminal Schwann cells within Meissner’s corpuscles. Pathogenic USH2A mutations cause Usher’s syndrome, associated with hearing loss and visual impairment. We show that patients with biallelic pathogenic USH2A mutations also have clear and specific impairments in vibrotactile touch perception, as do mutant mice lacking USH2A. Forepaw rapidly adapting mechanoreceptors innervating Meissner’s corpuscles, recorded from Ush2a−/− mice, showed large reductions in vibration sensitivity. However, the USH2A protein was not found in sensory neurons. Thus, loss of USH2A in corpuscular end-organs reduced mechanoreceptor sensitivity as well as vibration perception. Thus, a tether-like protein is required to facilitate detection of small-amplitude vibrations essential for the perception of fine-grained tactile surfaces.The present study was funded by grants from the Deutsche Forschungsgemeinshaft (grant nos. SFB665-B6 to G.R.L., SFB1315 to J.F.A.P. and SFB1158-A01 to S.G.L.) and grants from the European Research Council (grant nos. 789128 to G.R.L. and ERC-2015-CoG-682422 to J.F.A.P.). Additional funding was from the Institute of Health Carlos III (Spanish Ministry of Science and Innovation, grant no. FIS PI16/00539 to J.M.).Peer reviewe

Regulation of Adipocyte 11β-Hydroxysteroid Dehydrogenase Type 1 (11β-HSD1) by CCAAT/Enhancer-Binding Protein (C/EBP) β Isoforms, LIP and LAP

11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) catalyses intracellular regeneration of active glucocorticoids, notably in liver and adipose tissue. 11β-HSD1 is increased selectively in adipose tissue in human obesity, a change implicated in the pathogenesis of metabolic syndrome. With high fat (HF)-feeding, adipose tissue 11β-HSD1 is down-regulated in mice, plausibly to counteract metabolic disease. Transcription of 11β-HSD1 is directly regulated by members of the CCAAT/enhancer binding protein (C/EBP) family. Here we show that while total C/EBPβ in adipose tissue is unaltered by HF diet, the ratio of the C/EBPβ isoforms liver-enriched inhibitor protein (LIP) and liver-enriched activator protein (LAP) (C/EBPβ-LIP:LAP) is increased in subcutaneous adipose. This may cause changes in 11β-HSD1 expression since genetically modified C/EBPβ(+/L) mice, with increased C/EBPβ-LIP:LAP ratio, have decreased subcutaneous adipose 11β-HSD1 mRNA levels, whereas C/EBPβΔuORF mice, with decreased C/EBPβ-LIP:LAP ratio, show increased subcutaneous adipose 11β-HSD1. C/EBPβ-LIP:LAP ratio is regulated by endoplasmic reticulum (ER) stress and mTOR signalling, both of which are altered in obesity. In 3T3-L1 adipocytes, 11β-HSD1 mRNA levels were down-regulated following induction of ER stress by tunicamycin but were up-regulated following inhibition of mTOR by rapamycin. These data point to a central role for C/EBPβ and its processing to LIP and LAP in transcriptional regulation of 11β-HSD1 in adipose tissue. Down-regulation of 11β-HSD1 by increased C/EBPβ-LIP:LAP in adipocytes may be part of a nutrient-sensing mechanism counteracting nutritional stress generated by HF diet

Essential Requirement of CCAAT/Enhancer Binding Proteins in Embryogenesis

The CCAAT/enhancer binding proteins C/EBPα and C/EBPβ are related transcription factors that are important for the function of various organs in the postnatal mouse. Gene replacement and tissue culture experiments have suggested partial redundancy of both transcription factors. Here we show that mouse embryos deficient of both C/EBPα and C/EBPβ (C/EBPαβ(−/−)) die between embryonic day 10 (E10) and E11 and display defective placentas. In situ hybridization revealed that C/EBPα and C/EBPβ are coexpressed in the chorionic plate at E9.5 and later in the trophoblasts of the labyrinthine layer. In C/EBPαβ(−/−) placentas, allantoic blood vessels invaded the chorion; however, vessel expansion and development of the labyrinthine layer was impaired. Furthermore, a single copy of either C/EBPα in the absence of C/EBPβ or C/EBPβ in the absence of C/EBPα is sufficient to complete development, suggesting complementation of these C/EBPs during embryogenesis. A single copy of C/EBPα in the absence of C/EBPβ, however, fails to rescue survival after birth, suggesting haploinsufficiency of C/EBPα in newborns. Our data thus reveal novel essential, redundant, and dosage dependent functions of C/EBPs

Repression of Transcriptional Activity of C/EBPα by E2F-Dimerization Partner Complexes▿†

The transcription factor CCAAT/enhancer-binding protein α (C/EBPα) coordinates proliferation arrest and the differentiation of myeloid progenitors, adipocytes, hepatocytes, keratinocytes, and cells of the lung and placenta. C/EBPα transactivates lineage-specific differentiation genes and inhibits proliferation by repressing E2F-regulated genes. The myeloproliferative C/EBPα BRM2 mutant serves as a paradigm for recurrent human C-terminal bZIP C/EBPα mutations that are involved in acute myeloid leukemogenesis. BRM2 fails to repress E2F and to induce adipogenesis and granulopoiesis. The data presented here show that, independently of pocket proteins, C/EBPα interacts with the dimerization partner (DP) of E2F and that C/EBPα-E2F/DP interaction prevents both binding of C/EBPα to its cognate sites on DNA and transactivation of C/EBP target genes. The BRM2 mutant, in addition, exhibits enhanced interaction with E2F-DP and reduced affinity toward DNA and yet retains transactivation potential and differentiation competence that becomes exposed when E2F/DP levels are low. Our data suggest a tripartite balance between C/EBPα, E2F/DP, and pocket proteins in the control of proliferation, differentiation, and tumorigenesis

Multiple circadian oscillators in the photosensitive pike pineal gland : a study using organ and cell culture

International audienc

Photoreceptor cells of the pike pineal organ as cellular circadian oscillators

International audienc

Multiple circadian oscillators in the photosensitive pike pineal gland : a study using organ and cell culture

International audienc

C/EBPβΔuORF mice—a genetic model for uORF-mediated translational control in mammals

Upstream ORFs (uORFs) are translational control elements found predominantly in transcripts of key regulatory genes. No mammalian genetic model exists to experimentally validate the physiological relevance of uORF-regulated translation initiation. We report that mice deficient for the CCAAT/enhancer-binding protein β (C/EBPβ) uORF initiation codon fail to initiate translation of the autoantagonistic LIP (liver inhibitory protein) C/EBPβ isoform. C/EBPβΔuORF mice show hyperactivation of acute-phase response genes, persistent repression of E2F-regulated genes, delayed and blunted S-phase entry of hepatocytes after partial hepatectomy, and impaired osteoclast differentiation. These data and the widespread prevalence of uORFs in mammalian transcriptomes suggest a comprehensive role of uORF-regulated translation in (patho)physiology

Altered C/EBPβ-LIP:LAP ratio <i>in vivo</i> affects 11β-HSD1 expression in adipose tissue.

<p>(<b>A</b>) Representative western blot (40 µg protein/lane) showing levels of C/EBPβ-LAP (38 kDa LAP* +35 kDa LAP) and -LIP (20 kDa) isoforms in the adipose tissue of C/EBPβ mutants C/EBPβ^(+/L) (+/L), C/EBPβ^ΔuORF (ΔuORF) and control (Con) mice. Blots were stripped and reprobed with β-tubulin antibody, as loading control. All samples were analysed in the same gel but not all in adjacent lanes. (<b>B</b>) Real-time PCR measurement levels of mRNA encoding 11β-HSD1 in adipose tissue of wild type control mice (Con, white bar), C/EBPβ^(+/L) (+/L, black bar) and C/EBPβ^ΔuORF (ΔuORF, grey bar). C/EBPβ^(+/L) mice are heterozygous for an allele of C/EBPβ in which the normal gene has been replaced by C/EBPβ-LIP (a “knock-in”) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037953#pone.0037953-Smink1" target="_blank">[31]</a> and C/EBPβ^ΔuORF is homozygous for the deletion of upstream ORF codon <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037953#pone.0037953-Wethmar1" target="_blank">[32]</a>. Adipose 11β-HSD1 mRNA levels, normalized to TBP, are expressed relative to levels in control mice (arbitrarily set to 100%) and are mean±SEM; n = 6–9/group. *, p≤0.05; **, p<0.001.</p