scholarly journals Ets-2 Repressor Factor Silences Extrasynaptic Utrophin by N-Box–mediated Repression in Skeletal Muscle

2007 ◽  
Vol 18 (8) ◽  
pp. 2864-2872 ◽  
Author(s):  
Kelly J. Perkins ◽  
Utpal Basu ◽  
Murat T. Budak ◽  
Caroline Ketterer ◽  
Santhosh M. Baby ◽  
...  
Keyword(s):  
Promoter Activity ◽  
Molecular Mechanisms ◽  
Neuromuscular Junctions ◽  
Protein Product ◽  
Mrna Levels ◽  
Laser Capture Microscopy ◽  
Erk Phosphorylation ◽  
Duchenne's Muscular Dystrophy

Utrophin is the autosomal homologue of dystrophin, the protein product of the Duchenne's muscular dystrophy (DMD) locus. Utrophin expression is temporally and spatially regulated being developmentally down-regulated perinatally and enriched at neuromuscular junctions (NMJs) in adult muscle. Synaptic localization of utrophin occurs in part by heregulin-mediated extracellular signal-regulated kinase (ERK)-phosphorylation, leading to binding of GABPα/β to the N-box/EBS and activation of the major utrophin promoter-A expressed in myofibers. However, molecular mechanisms contributing to concurrent extrasynaptic silencing that must occur to achieve NMJ localization are unknown. We demonstrate that the Ets-2 repressor factor (ERF) represses extrasynaptic utrophin-A in muscle. Gel shift and chromatin immunoprecipitation studies demonstrated physical association of ERF with the utrophin-A promoter N-box/EBS site. ERF overexpression repressed utrophin-A promoter activity; conversely, small interfering RNA-mediated ERF knockdown enhanced promoter activity as well as endogenous utrophin mRNA levels in cultured muscle cells in vitro. Laser-capture microscopy of tibialis anterior NMJ and extrasynaptic transcriptomes and gene transfer studies provide spatial and direct evidence, respectively, for ERF-mediated utrophin repression in vivo. Together, these studies suggest “repressing repressors” as a potential strategy for achieving utrophin up-regulation in DMD, and they provide a model for utrophin-A regulation in muscle.

scholarly journals Estrogen Up-Regulates Hepatic Expression of Suppressors of Cytokine Signaling-2 and -3 in Vivo and in Vitro

Endocrinology ◽  
2004 ◽  
Vol 145 (12) ◽  
pp. 5525-5531 ◽  
Author(s):  
Gary M. Leong ◽  
Sofia Moverare ◽  
Jesena Brce ◽  
Nathan Doyle ◽  
Klara Sjögren ◽  
...  
Keyword(s):  
Promoter Activity ◽  
Hepatoma Cells ◽  
Cytokine Signaling ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Wild Type ◽  
Hepatic Expression ◽  
Suppressors Of Cytokine Signaling

Abstract Suppressors of cytokine signaling (SOCS) are important negative regulators of cytokine action. We recently reported that estrogen stimulates SOCS-2 expression and inhibits GH signaling in kidney cells. The effects of estrogen on SOCS expression in other tissues are unclear. The aim of this study was to investigate in vivo and in vitro whether estrogen affected SOCS expression in the liver, a major target organ of GH. The in vivo hepatic effects of estrogen on ovariectomized mice lacking estrogen receptor (ER)-α, ERβ, or both and their wild-type littermates were examined by DNA microarray analysis. In vitro, the effects of estrogen on SOCS expression in human hepatoma cells were examined by reverse transcription quantitative PCR. Long-term (3 wk) estrogen treatment induced a 2- to 3-fold increase in hepatic expression of SOCS-2 and -3 in wild-type and ERβ knockout mice but not in those lacking ERα or both ER subtypes. Short-term treatment (at 24 h) increased the mRNA level of SOCS-3 but not SOCS-2. In cultured hepatoma cells, estrogen increased SOCS-2 and -3 mRNA levels by 2-fold in a time- and dose-dependent manner (P < 0.05). Estrogen induced murine SOCS-3 promoter activity by 2-fold (P < 0.05) in constructs containing a region between nucleotides −1862 and −855. Moreover, estrogen and GH had additive effects on the SOCS-3 promoter activity. In summary, estrogen, via ERα, up-regulated hepatic expression of SOCS-2 and -3, probably through transcriptional activation. This indicates a novel mechanism of estrogen regulation of cytokine action.

scholarly journals Novel insights into the regulation of chemerin expression: role of acute-phase cytokines and DNA methylation

2019 ◽  
Author(s):  
Kamila Kwiecien ◽  
Piotr Brzoza ◽  
Pawel Majewski ◽  
Izabella Skulimowska ◽  
Kamil Bednarczyk ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Acute Phase ◽  
Molecular Mechanisms ◽  
Retinoic Acid Receptor ◽  
Constitutive Expression ◽  
Sequencing Analysis ◽  
Mrna Levels ◽  
Pleiotropic Actions

AbstractChemerin is a chemoattractant protein with adipokine properties encoded by the retinoic acid receptor responder 2 (RARRES2) gene. It has gained more attention over the past few years due to its multilevel impact on metabolism and immune responses. The pleiotropic actions of chemerin include chemotaxis of dendritic cells, macrophages and natural killers (NK) subsets, bactericidal activity as well as regulation of adipogenesis and glucose metabolism. Therefore, reflecting the pleiotropic actions of chemerin, expression of RARRES2 is regulated by a variety of inflammatory and metabolic mediators. However, for most cell types, the molecular mechanisms controlling constitutive and regulated chemerin expression are poorly characterized. Here we show that RARRES2 mRNA levels in murine adipocytes are upregulated in vitro and in vivo by acute-phase cytokines, IL-1β and OSM. In contrast to adipocytes, these cytokines exerted a weak, if any, response in mouse hepatocytes, suggesting that the effect of IL-1β and OSM on chemerin expression is specific to fat tissue. Moreover, we show that DNA methylation controls the constitutive expression of chemerin. Bisulfite sequencing analysis showed low methylation levels within −735 to +258 bp of the murine RARRES2 gene promoter in unstimulated adipocytes and hepatocytes. In contrast to these cells, the RARRES2 promoter is highly methylated in B lymphocytes, cells that do not produce chemerin. Together, our findings reveal previously uncharacterized mediators and mechanisms controlling chemerin expression in various cells.

scholarly journals Building neuromuscular junctions in vitro

Development ◽  
2020 ◽  
Vol 147 (22) ◽  
pp. dev193920
Author(s):  
Susie Barbeau ◽  
Julie Tahraoui-Bories ◽  
Claire Legay ◽  
Cécile Martinat
Keyword(s):  
Human Disease ◽  
Molecular Mechanisms ◽  
Neuromuscular Junctions ◽  
Ex Vivo ◽  
Culture Methods ◽  
Recent Developments ◽  
Chemical Synapses ◽  
And Function

ABSTRACTThe neuromuscular junction (NMJ) has been the model of choice to understand the principles of communication at chemical synapses. Following groundbreaking experiments carried out over 60 years ago, many studies have focused on the molecular mechanisms underlying the development and physiology of these synapses. This Review summarizes the progress made to date towards obtaining faithful models of NMJs in vitro. We provide a historical approach discussing initial experiments investigating NMJ development and function from Xenopus to mice, the creation of chimeric co-cultures, in vivo approaches and co-culture methods from ex vivo and in vitro derived cells, as well as the most recent developments to generate human NMJs. We discuss the benefits of these techniques and the challenges to be addressed in the future for promoting our understanding of development and human disease.

scholarly journals Coordinate Transcription of the ADAMTS-1 Gene by Luteinizing Hormone and Progesterone Receptor

2004 ◽  
Vol 18 (10) ◽  
pp. 2463-2478 ◽  
Author(s):  
Kari M. H. Doyle ◽  
Darryl L. Russell ◽  
Venkataraman Sriraman ◽  
JoAnne S. Richards
Keyword(s):  
Progesterone Receptor ◽  
Granulosa Cells ◽  
Binding Sites ◽  
Promoter Activity ◽  
Molecular Mechanisms ◽  
Luciferase Reporter ◽  
Nuclear Factor ◽  
Nuclear Factor 1

Abstract ADAMTS-1 (a disintegrin and metalloproteinase with thrombospondin-like motifs) is a multifunctional protease that is expressed in periovulatory follicles. Herein we show that induction of ADAMTS-1 message in vivo and transcription of the ADAMTS-1 promoter in cultured granulosa cells are dependent on separable but coordinate actions of LH and the progesterone receptor (PR). To analyze the molecular mechanisms by which LH and PR regulate this gene, truncations and site-specific mutants of ADAMTS-1 promoter-luciferase reporter constructs (ADAMTS-1-Luc) were generated and transfected into rat granulosa cell cultures. Three regions of the promoter were found to be important for basal activity, two of which were guanine cytosine-rich binding sites for specificity proteins Sp1/Sp3 and the third bound a nuclear factor 1-like factor. Despite the absence of a consensus PR DNA response element in the proximal ADAMTS-1 promoter, cotransfection of a PRA (or PRB) expression vector stimulated ADAMTS-1 promoter activity, a response that was reduced by the PR antagonist ZK98299. Forskolin plus phorbol myristate acetate also increased promoter activity and, when added to cells cotransfected with PRA, ADAMTS-1 promoter activity increased further. Activation of the ADAMTS-1 promoter by PRA involves functional CAAT enhancer binding protein β, nuclear factor 1-like factor, and three Sp1/Sp3 binding sites as demonstrated by transfection of mutated promoter constructs. In summary, LH and PRA/B exert distinct but coordinate effects on transactivation of the ADAMTS-1 gene in granulosa cells in vivo and in vitro with PR acting as an inducible coregulator of the ADAMTS-1 gene.

scholarly journals Lactobacillus acidophilus stimulates the expression of SLC26A3 via a transcriptional mechanism

2010 ◽  
Vol 298 (3) ◽  
pp. G395-G401 ◽  
Author(s):  
Geetu Raheja ◽  
Varsha Singh ◽  
Ke Ma ◽  
Redouane Boumendjel ◽  
Alip Borthakur ◽  
...  
Keyword(s):  
Western Blot ◽  
Lactobacillus Acidophilus ◽  
Promoter Activity ◽  
In Vivo Studies ◽  
Mrna Levels ◽  
Long Term Effects ◽  
Exchange Activity

Clinical efficacy of probiotics in treating various forms of diarrhea has been clearly established. However, mechanisms underlying antidiarrheal effects of probiotics are not completely defined. Diarrhea is caused either by decreased absorption or increased secretion of electrolytes and solutes in the intestine. In this regard, the electroneutral absorption of two major electrolytes, Na+ and Cl−, occurs mainly through the coupled operation of Na+/H+ exchangers and Cl−/OH− exchangers. Previous studies from our laboratory have shown that Lactobacillus acidophilus (LA) acutely stimulated Cl−/OH− exchange activity via an increase in the surface levels of the apical anion exchanger SLC26A3 (DRA). However, whether probiotics influence SLC26A3 expression and promoter activity has not been examined. The present studies were, therefore, undertaken to investigate the long-term effects of LA on SLC26A3 expression and promoter activity. Treatment of Caco-2 cells with LA for 6–24 h resulted in a significant increase in Cl−/OH− exchange activity. DRA mRNA levels were also significantly elevated in response to LA treatment starting as early as 8 h. Additionally, the promoter activity of DRA was increased by more than twofold following 8 h LA treatment of Caco-2 cells. Similar to the in vitro studies, in vivo studies using mice gavaged with LA also showed significantly increased DRA mRNA (∼4-fold) and protein expression in the colonic regions as assessed by Western blot analysis and immunofluorescence. In conclusion, increase in DRA promoter activity and expression may contribute to the upregulation of intestinal electrolyte absorption and might underlie the potential antidiarrheal effects of LA.

scholarly journals Calcineurin activates interleukin-6 transcription in mouse skeletal muscle in vivo and in C2C12 myotubes in vitro

2010 ◽  
Vol 298 (1) ◽  
pp. R198-R210 ◽  
Author(s):  
David L. Allen ◽  
Jill J. Uyenishi ◽  
Allison S. Cleary ◽  
Ryan S. Mehan ◽  
Sarah F. Lindsay ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Endurance Exercise ◽  
Binding Sites ◽  
Promoter Activity ◽  
Wheel Running ◽  
Mrna Levels ◽  
Dna Binding Sites ◽  
Single Bout

Expression of the cytokine interleukin-6 (IL-6) by skeletal muscle is hugely increased in response to a single bout of endurance exercise, and this appears to be mediated by increases in intracellular calcium. We examined the effects of endurance exercise on IL-6 mRNA levels and promoter activity in skeletal muscle in vivo, and the role of the calcium-activated calcineurin signaling pathway on muscle IL-6 expression in vivo and in vitro. IL-6 mRNA levels in the mouse tibialis anterior (TA) were increased 2–10-fold by a single bout of treadmill exercise or by 3 days of voluntary wheel running. Moreover, an IL-6 promoter-driven luciferase transgene was activated in TA by both treadmill and wheel-running exercise and by injection with a calcineurin plasmid. Exercise also increased muscle mRNA expression of the calcineurin regulatory gene MCIP1, as did treatment of C2C12 myotubes with the calcium ionophore A23187. Cotransfection of C2C12 myotubes with a constitutively active calcineurin construct significantly increased while cotransfection with the calcineurin inhibitor CAIN inhibited activity of a mouse IL-6 promoter-reporter construct. Cotransfection with a myocyte enhancer-factor-2 (MEF-2) expression construct increased basal IL-6 promoter activity and augmented the effects of calcineurin cotransfection, while cotransfection with the MEF-2 antagonist MITR repressed calcineurin-activated IL-6 promoter activity in vitro. Surprisingly, cotransfection with a dominant-negative form of another calcineurin-activated transcription factor, nuclear factor activator of T cells (NFAT), greatly potentiated both basal and calcineurin-stimulated IL-6 promoter activity in C2C12 myotubes. Mutation of the MEF-2 DNA binding sites attenuated, while mutation of the NFAT DNA binding sites potentiated basal and calcineurin-activated IL-6 promoter activity. Finally, CREB and C/EBP were necessary for basal IL-6 promoter activity and sufficient to increase IL-6 promoter activity but had minimal roles in calcineurin-activated IL-6 promoter activity. Together, these results suggest that IL-6 transcription in skeletal muscle cells can be activated by a calcineurin-MEF-2 axis which is antagonized by NFAT.

scholarly journals Effects of Rexinoids on Thyrotrope Function and the Hypothalamic-Pituitary-Thyroid Axis

Endocrinology ◽  
2006 ◽  
Vol 147 (3) ◽  
pp. 1438-1451 ◽  
Author(s):  
Vibha Sharma ◽  
William R. Hays ◽  
William M. Wood ◽  
Umarani Pugazhenthi ◽  
Donald L. St. Germain ◽  
...  
Keyword(s):  
Direct Effect ◽  
Promoter Activity ◽  
Retinoic Acid Receptor ◽  
Mrna Levels ◽  
Central Hypothyroidism ◽  
Iodothyronine Deiodinase ◽  
Pituitary Thyroid Axis ◽  
Thyroid Axis

Retinoid X receptor (RXR)-selective retinoids (rexinoids) can cause central hypothyroidism in humans, and this effect has been confirmed in rodent models. In this report, we characterized the effect of rexinoids on the hypothalamic-pituitary-thyroid axis in mice and TSH regulation in a thyrotrope-derived cell line. The synthetic rexinoid (LG 268) suppressed TSH and T4 levels in mice. Hypothalamic TRH mRNA was unaffected, but steady-state pituitary TSHβ mRNA levels were significantly lowered, suggesting a direct effect of rexinoids on thyrotropes. LG 268 suppressed TSH protein secretion and TSHβ mRNA in TαT1 thyrotropes as early as 8 h after treatment, whereas the retinoic acid receptor-selective retinoid (TTNPB) had no effect. Type 2 iodothyronine deiodinase (D2) mRNA and activity were suppressed by LG 268 in TαT1 cells, whereas only D2 mRNA was suppressed in mouse pituitaries. LG 268 suppressed TSHβ promoter activity by 42% and the −200 to −149 region accounted for a majority of the LG 268-mediated suppression of promoter activity. The RXRγ isotype is expressed in thyrotropes. In vitro transfection and in vivo transgenic studies indicate that any RXR isotype can mediate TSH suppression by rexinoids, but the RXRγ isotype is most efficient at mediating this response. RXRγ-deficient mice lacked pituitary D2 mRNA suppression by LG 268, but D2 activity remained intact. In summary, RXR-selective retinoids (rexinoids) have multiple effects on the hypothalamic-pituitary-thyroid axis. Rexinoids directly suppress TSH secretion, TSHβ mRNA levels and promoter activity, and D2 mRNA levels but have no direct effect on hypothalamic TRH levels. Rexinoids also stimulate type 1 iodothyronine deiodinase activity in the liver and pituitary.

scholarly journals Donepezil Regulates LPS and Aβ-Stimulated Neuroinflammation through MAPK/NLRP3 Inflammasome/STAT3 Signaling

2021 ◽  
Vol 22 (19) ◽  
pp. 10637
Author(s):  
Jieun Kim ◽  
Hyun-ju Lee ◽  
Seon Kyeong Park ◽  
Jin-Hee Park ◽  
Ha-Ram Jeong ◽  
...  
Keyword(s):  
Nlrp3 Inflammasome ◽  
Molecular Mechanisms ◽  
Acetylcholinesterase Inhibitors ◽  
Mapk Signaling ◽  
Inos Mrna ◽  
Mrna Levels ◽  
Stat3 Phosphorylation ◽  
5Xfad Mice

The acetylcholinesterase inhibitors donepezil and rivastigmine have been used as therapeutic drugs for Alzheimer’s disease (AD), but their effects on LPS- and Aβ-induced neuroinflammatory responses and the underlying molecular pathways have not been studied in detail in vitro and in vivo. In the present study, we found that 10 or 50 μM donepezil significantly decreased the LPS-induced increases in the mRNA levels of a number of proinflammatory cytokines in BV2 microglial cells, whereas 50 μM rivastigmine significantly diminished only LPS-stimulated IL-6 mRNA levels. In subsequent experiments in primary astrocytes, donepezil suppressed only LPS-stimulated iNOS mRNA levels. To identify the molecular mechanisms by which donepezil regulates LPS-induced neuroinflammation, we examined whether donepezil alters LPS-stimulated proinflammatory responses by modulating LPS-induced downstream signaling and the NLRP3 inflammasome. Importantly, we found that donepezil suppressed LPS-induced AKT/MAPK signaling, the NLRP3 inflammasome, and transcription factor NF-kB/STAT3 phosphorylation to reduce neuroinflammatory responses. In LPS-treated wild-type mice, a model of neuroinflammatory disease, donepezil significantly attenuated LPS-induced microglial activation, microglial density/morphology, and proinflammatory cytokine COX-2 and IL-6 levels. In a mouse model of AD (5xFAD mice), donepezil significantly reduced Aβ-induced microglial and astrocytic activation, density, and morphology. Taken together, our findings indicate that donepezil significantly downregulates LPS- and Aβ-evoked neuroinflammatory responses in vitro and in vivo and may be a therapeutic agent for neuroinflammation-associated diseases such as AD.

In vitro and in vivo characterization of the minimal promoter region of the human thiamin transporter SLC19A2

2003 ◽  
Vol 285 (3) ◽  
pp. C633-C641 ◽  
Author(s):  
Jack C. Reidling ◽  
Hamid M. Said
Keyword(s):  
Transgenic Mice ◽  
Mammalian Cells ◽  
Promoter Activity ◽  
Molecular Mechanisms ◽  
Regulatory Region ◽  
Minimal Promoter ◽  
Human Tissues ◽  
Cis Elements

The molecular mechanisms involved in the regulation of thiamin transport in mammalian cells are poorly understood. Previous studies established that a human thiamin transporter, SLC19A2, plays a role in thiamin uptake in human tissues. We cloned the 5′ regulatory region of the SLC19A2 gene, identified the minimal promoter required for basal activity, and located multiple putative cis elements. To further characterize the SLC19A2 promoter, we investigated, in the present study, the role of the putative cis elements in regulating the activity of the SLC19A2 promoter in vitro and confirmed the activity of the SLC19A2 promoter in vivo. In vitro studies demonstrated that mutation of specific cis elements in the SLC19A2 minimal promoter [Gut-enriched Krupple-like factor (GKLF), nuclear factor-1 (NF-1), and stimulating protein-1 (SP-1)] led to a decrease in activity. Using electrophoretic mobility shift assays, four specific DNA/protein complexes were identified. The interacting factors were determined by oligonucleotide competition and antibody supershift analysis and shown to be GKLF, NF-1, and SP-1. Cotransfection studies of the SLC19A2 promoter with an SP-1 containing vector in Drosophila SL2 cells further confirmed a role for SP-1 in regulating SLC19A2 promoter activity. In vivo studies using transgenic mice established the functionality of the full-length and minimal SLC19A2 promoters. Furthermore, our studies revealed that the pattern of expression of the SLC19A2 promoter-Luciferase constructs in transgenic mice was similar to the reported SLC19A2 RNA expression pattern in native human tissues. The results demonstrate the importance of GKLF, NF-1, and SP-1 in regulating the activity of the SLC19A2 promoter and provide direct in vivo confirmation of promoter activity.

In Vitro Studies of Acrylamide Neurotoxicity in Rat Pheochromocytoma (PC12) Cells

1996 ◽  
Vol 24 (3) ◽  
pp. 359-366
Author(s):  
Weiquan W. Lin ◽  
Larry R. Johnson ◽  
Marvin A. Friedman ◽  
Mohamed B. Abou-Donia
Keyword(s):  
Protein Synthesis ◽  
Pc12 Cells ◽  
Molecular Mechanisms ◽  
Mrna Levels ◽  
Neurofilament Protein ◽  
Ngf Receptor ◽  
Pheochromocytoma Pc12 ◽  
Axonal Swellings

This review discusses our studies on molecular mechanisms of acrylamide neurotoxicity by using the rat pheochromocytoma (PC12) cell line. The results showed that: a) acrylamide altered the gross morphology of PC12 cells; b) acrylamide induced neurofilament accumulation in PC12 cells; c) the effects of acrylamide on PC12 cells are consistent with its neurotoxicity in vivo; d) acrylamide stimulated neurofilament protein synthesis in PC12 cells; e) acrylamide did not act via nerve growth factor (NGF) receptor gp140trk to regulate neurofilament synthesis in PC12 cells; f) dexamethasone antagonised NGF and/or acrylamide-induced neurofilament protein synthesis and expression; and g) acrylamide differentially regulated the mRNA levels of three neurofilament subunit genes in PC12 cells. These molecular studies provide the first evidence that: a) there are distinctive and convergent signalling pathways for NGF-regulated and acrylamide-regulated neurofilament expression; b) acrylamide may differentially regulate the expression of each subunit, resulting in aberrant accumulation of neurofilament proteins; and c) there is a dexamethasone-sensitive signalling step common to NGF and acrylamide. These results could partially explain the mechanisms of neurofilament accumulation in distal axonal swellings, a pathognomonic feature of acrylamide neurotoxicity.

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