Human Osteogenesis Involves Differentiation-Dependent Increases in the Morphogenically Active 3′ Alternative Splicing Variant of Acetylcholinesterase

ABSTRACT The extended human acetylcholinesterase (AChE) promoter contains many binding sites for osteogenic factors, including 1,25-(OH)2 vitamin D3 and 17β-estradiol. In differentiating osteosarcoma Saos-2 cells, both of these factors enhanced transcription of the AChE mRNA variant 3′ terminated with exon 6 (E6-AChE mRNA), which encodes the catalytically and morphogenically active E6-AChE isoform. In contrast, antisense oligodeoxynucleotide suppression of E6-AChE mRNA expression increased Saos-2 proliferation in a dose- and sequence-dependent manner. The antisense mechanism of action was most likely mediated by mRNA destruction or translational arrest, as cytochemical staining revealed reduction in AChE gene expression. In vivo, we found that E6-AChE mRNA levels rose following midgestation in normally differentiating, postproliferative fetal chondrocytes but not in the osteogenically impaired chondrocytes of dwarf fetuses with thanatophoric dysplasia. Taken together, these findings suggest morphogenic involvement of E6-AChE in the proliferation-differentiation balance characteristic of human osteogenesis.

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Glucolipotoxicity and GLP-1 secretion

BMJ Open Diabetes Research & Care ◽

10.1136/bmjdrc-2020-001905 ◽

2021 ◽

Vol 9 (1) ◽

pp. e001905

Author(s):

Jung-Hee Hong ◽

Dae-Hee Kim ◽

Moon-Kyu Lee

Keyword(s):

Glucose Transporter ◽

Cell Function ◽

Nicotinamide Adenine Dinucleotide Phosphate ◽

Mrna Levels ◽

Dependent Manner ◽

Gene Expressions ◽

Simultaneous Treatment ◽

L Cells ◽

Triglyceride Accumulation

IntroductionThe concept of glucolipotoxicity refers to the combined, deleterious effects of elevated glucose and/or fatty acid levels.Research design and methodsTo investigate the effects of chronic glucolipotoxicity on glucagon-like peptide-1-(7-36) amide (GLP-1) secretion, we generated glucolipotoxic conditions in human NCI-H716 enteroendocrine cells using either 5 or 25 mM glucose with or without 500 µM palmitate for 72 hours. For in vivo study, we have established a chronic nutrient infusion model in the rat. Serial blood samples were collected for 2 hours after the consumption of a mixed meal to evaluate insulin sensitivity and β-cell function.ResultsChronic glucolipotoxic conditions decreased GLP-1 secretion and the expressions of pCREB, pGSK3β, β-catenin, and TCF7L2 in NCI-H716 cells. Glucolipotoxicity conditions reduced glucose transporter expression, glucose uptake, and nicotinamide-adenine dinucleotide phosphate (NADPH) levels in L-cells, and increased triglyceride accumulation. In contrast, PPARα and ATP levels were reduced, which correlated well with decreased levels of SUR1 and Kir6.2, cAMP contents and expressions of pCAMK2, EPAC and PKA. We also observed an increase in reactive oxygen species production, UCP2 expression and Complex I activity. Simultaneous treatment with insulin restored the GLP-1 secretion. Glucolipotoxic conditions decreased insulin secretion in a time-dependent manner in INS-1 cells, which was recovered with exendin-4 cotreatment. Glucose and SMOFlipid infusion for 6 hours decreased GLP-1 secretion and proglucagon mRNA levels as well as impaired the glucose tolerance, insulin and C-peptide secretion in rats.ConclusionThese results provide evidence for the first time that glucolipotoxicity could affect GLP-1 secretion through changes in glucose and lipid metabolism, gene expressions, and proglucagon biosynthesis and suggest the interrelationship between glucolipotoxicities of L-cells and β-cells which develops earlier than that of L-cells.

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

Endocrinology ◽

10.1210/en.2004-0061 ◽

2004 ◽

Vol 145 (12) ◽

pp. 5525-5531 ◽

Cited By ~ 50

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.

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Tumor-Stroma Crosstalk Enhances REG3A Expressions that Drive the Progression of Hepatocellular Carcinoma

International Journal of Molecular Sciences ◽

10.3390/ijms21020472 ◽

2020 ◽

Vol 21 (2) ◽

pp. 472 ◽

Cited By ~ 2

Author(s):

Yuri Cho ◽

Min Ji Park ◽

Koeun Kim ◽

Jae-Young Park ◽

Jihye Kim ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Microarray Analysis ◽

Cdna Microarray ◽

Tumor Stroma ◽

Mrna Levels ◽

Dependent Manner ◽

Cdna Microarray Analysis ◽

Hcc Cells

Abstract: Background: Crosstalk between tumors and their microenvironment plays a crucial role in the progression of hepatocellular carcinoma (HCC). However, there is little existing information about the key signaling molecule that modulates tumor-stroma crosstalk. Methods: Complementary DNA (cDNA) microarray analysis was performed to identify the key molecule in tumor-stroma crosstalk. Subcutaneous xenograft in vivo murine model, immunoblotting, immunofluorescence, and real-time polymerase chain reaction using HCC cells and tissues were performed. Results: The key molecule, regenerating gene protein-3A (REG3A), was most significantly enhanced when coculturing HCC cells and activated human hepatic stellate cells (HSCs) (+8.2 log) compared with monoculturing HCC cells using cDNA microarray analysis. Downregulation of REG3A using small interfering RNA significantly decreased the proliferation of HSC-cocultured HCC cells in vitro and in vivo, and enhanced deoxycholic acid-induced HCC cell apoptosis. Crosstalk-induced REG3A upregulation was modulated by platelet-derived growth factor ββ (PDGF-ββ) in p42/44-dependent manner. REG3A mRNA levels in human HCC tissues were upregulated 1.8-fold compared with non-tumor tissues and positively correlated with PDGF-ββ levels. Conclusions: REG3A/p42/44 pathway/PDGF-ββ signaling plays a significant role in hepatocarcinogenesis via tumor-stroma crosstalk. Targeting REG3A is a potential novel therapeutic target for the management of HCCs by inhibiting crosstalk between HCC cells and HSCs.

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Suppression of the GnRH Pulse Generator by Neurokinin B Involves a κ-Opioid Receptor-Dependent Mechanism

Endocrinology ◽

10.1210/en.2012-1574 ◽

2012 ◽

Vol 153 (10) ◽

pp. 4894-4904 ◽

Cited By ~ 53

Author(s):

P. Grachev ◽

X. F. Li ◽

J. S. Kinsey-Jones ◽

A. L. di Domenico ◽

R. P. Millar ◽

...

Keyword(s):

Opioid Receptor ◽

Pulse Generator ◽

Pulse Frequency ◽

Mrna Levels ◽

Dependent Manner ◽

Neurokinin B ◽

Kndy Neurons ◽

Lh Secretion ◽

Dose Dependent

Abstract Neurokinin B (NKB) and its receptor (NK3R) are coexpressed with kisspeptin, Dynorphin A (Dyn), and their receptors [G-protein-coupled receptor-54 (GPR54)] and κ-opioid receptor (KOR), respectively] within kisspeptin/NKB/Dyn (KNDy) neurons in the hypothalamic arcuate nucleus (ARC), the proposed site of the GnRH pulse generator. Much previous research has employed intracerebroventricular (icv) administration of KNDy agonists and antagonists to address the functions of KNDy neurons. We performed a series of in vivo neuropharmacological experiments aiming to determine the role of NKB/NK3R signaling in modulating the GnRH pulse generator and elucidate the interaction between KNDy neuropeptide signaling systems, targeting our interventions to ARC KNDy neurons. First, we investigated the effect of intra-ARC administration of the selective NK3R agonist, senktide, on pulsatile LH secretion using a frequent automated serial sampling method to obtain blood samples from freely moving ovariectomized 17β-estradiol-replaced rats. Our results show that senktide suppresses LH pulses in a dose-dependent manner. Intra-ARC administration of U50488, a selective KOR agonist, also caused a dose-dependent, albeit more modest, decrease in LH pulse frequency. Thus we tested the hypothesis that Dyn/KOR signaling localized to the ARC mediates the senktide-induced suppression of the LH pulse by profiling pulsatile LH secretion in response to senktide in rats pretreated with nor-binaltorphimine, a selective KOR antagonist. We show that nor-binaltorphimine blocks the senktide-induced suppression of pulsatile LH secretion but does not affect LH pulse frequency per se. In order to address the effects of acute activation of ARC NK3R, we quantified (using quantitative RT-PCR) changes in mRNA levels of KNDy-associated genes in hypothalamic micropunches following intra-ARC administration of senktide. Senktide down-regulated expression of genes encoding GnRH and GPR54 (GNRH1 and Kiss1r, respectively), but did not affect the expression of Kiss1 (which encodes kisspeptin). We conclude that NKB suppresses the GnRH pulse generator in a KOR-dependent fashion and regulates gene expression in GnRH neurons.

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Comparison of the Effects on Reversal of Multi-Drug Resistance of 5-Bromotetrandrine and Tetrandrine in K562/A02 Cell Line in Vivo and in Vitro

Blood ◽

10.1182/blood.v112.11.5059.5059 ◽

2008 ◽

Vol 112 (11) ◽

pp. 5059-5059

Author(s):

Bao-An Chen ◽

Jue-qiong Wang ◽

Jian Cheng ◽

Feng Gao ◽

Wen-lin Xu ◽

...

Keyword(s):

Cell Line ◽

Nude Mice ◽

Leukemia Cell Line ◽

Multi Drug Resistance ◽

Human Leukemia ◽

Mrna Levels ◽

Dependent Manner ◽

Intracellular Accumulation

Abstract Objective This study was to compare the reversal effect of 5-bromotetrandrine (BrTet) with Tetrandrine (Tet) when combined with ADM on multidrug resistance cell line K562/A02 and to investigate the reversal mechanism of this new derivative. Methods The protein levels of P-glycoprotein (P-gp) were detected by fluorospectrophotometry and Western blot. The mRNA levels of P-gp were determined by RT-PCR. The in vivo effect of Tet was investigated using nude mice grafted with sensitive human leukemia cell line K562 and MDR cell line K562/A02. Results Flow cytometry assay showed that 1.0 μMol/L BrTet significantly increased the apoptosis percentage. BrTet also enhanced the intracellular accumulation of ADM in K562/A02 cells and its potency was greater than that of Tet at the same concentrations. BrTet inhibited the overexpression of P-gp and down regulated MDR1 mRNA expression in K562/A02 cells in a dose-dependent manner. In nude mice bearing K562 xenografts on the left flank and K562/A02 xenografts on the right flank, i.p. injection of 10 mg/kg BrTet significantly enhanced the antitumor activity of ADM against K562/A02 xenografts with inhibitory rates of 26.1%, while ADM alone inhibited the growth of KBv200 xenografts by only 5.8%. Conclusion BrTet showed significant MDR reversal activity in vitro and in vivo. Its activity may be related to the inhibition of P-gp overexpression and the increase in intracellular accumulation of anticancer drugs, which lead to more K562/A02 cells apoptosis.

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Liganded Thyroid Hormone Receptor Induces Nucleosome Removal and Histone Modifications to Activate Transcription during Larval Intestinal Cell Death and Adult Stem Cell Development

Endocrinology ◽

10.1210/en.2011-1736 ◽

2012 ◽

Vol 153 (2) ◽

pp. 961-972 ◽

Cited By ~ 27

Author(s):

Kazuo Matsuura ◽

Kenta Fujimoto ◽

Liezhen Fu ◽

Yun-Bo Shi

Keyword(s):

Cell Death ◽

Gene Regulation ◽

Thyroid Hormone ◽

Chromatin Remodeling ◽

Histone Modifications ◽

Intestinal Cell ◽

Mrna Levels ◽

Dependent Manner ◽

Vertebrate Development

Thyroid hormone (T3) plays an important role in regulating multiple cellular and metabolic processes, including cell proliferation, cell death, and energy metabolism, in vertebrates. Dysregulation of T3 signaling results in developmental abnormalities, metabolic defects, and even cancer. We used T3-dependent Xenopus metamorphosis as a model to study how T3 regulates transcription during vertebrate development. T3 exerts its metamorphic effects through T3 receptors (TR). TR recruits, in a T3-dependent manner, cofactor complexes that can carry out chromatin remodeling/histone modifications. Whether and how histone modifications change upon gene regulation by TR during vertebrate development is largely unknown. Here we analyzed histone modifications at T3 target genes during intestinal metamorphosis, a process that involves essentially total apoptotic degeneration of the simple larval epithelium and de novo development of the adult epithelial stem cells, followed by their proliferation and differentiation into the complex adult epithelium. We demonstrated for the first time in vivo during vertebrate development that TR induces the removal of core histones at the promoter region and the recruitment of RNA polymerase. Furthermore, a number of histone activation and repression marks have been defined based on correlations with mRNA levels in cell cultures. Most but not all correlate with gene expression induced by liganded TR during development, suggesting that tissue and developmental context influences the roles of histone modifications in gene regulation. Our findings provide important mechanistic insights on how chromatin remodeling affects developmental gene regulation in vivo.

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Erythropoietin mediates hepcidin expression in hepatocytes through EPOR signaling and regulation of C/EBPα

Blood ◽

10.1182/blood-2007-08-106195 ◽

2008 ◽

Vol 111 (12) ◽

pp. 5727-5733 ◽

Cited By ~ 158

Author(s):

Jorge P. Pinto ◽

Sara Ribeiro ◽

Helena Pontes ◽

Shifaan Thowfeequ ◽

David Tosh ◽

...

Keyword(s):

Transcription Factor ◽

Cell Model ◽

Transcriptional Response ◽

In Vivo Studies ◽

Systemic Absorption ◽

Mrna Levels ◽

Dependent Manner ◽

Hepcidin Expression ◽

Factor C

Abstract Hepcidin is the principal iron regulatory hormone, controlling the systemic absorption and remobilization of iron from intracellular stores. Recent in vivo studies have shown that hepcidin is down-regulated by erythropoiesis, anemia, and hypoxia, which meets the need of iron input for erythrocyte production. Erythropoietin (EPO) is the primary signal that triggers erythropoiesis in anemic and hypoxic conditions. Therefore, a direct involvement of EPO in hepcidin regulation can be hypothesized. We report here the regulation of hepcidin expression by EPO, in a dose-dependent manner, in freshly isolated mouse hepatocytes and in the HepG2 human hepatocyte cell model. The effect is mediated through EPOR signaling, since hepcidin mRNA levels are restored by pretreatment with an EPOR-blocking antibody. The transcription factor C/EBPα showed a pattern of expression similar to hepcidin, at the mRNA and protein levels, following EPO and anti-EPOR treatments. Chromatin immunoprecipitation experiments showed a significant decrease of C/EBPα binding to the hepcidin promoter after EPO supplementation, suggesting the involvement of this transcription factor in the transcriptional response of hepcidin to EPO.

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Antiobesity Effects of UnripeRubus coreanusMiquel and Its Constituents: AnIn VitroandIn VivoCharacterization of the Underlying Mechanism

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2016/4357656 ◽

2016 ◽

Vol 2016 ◽

pp. 1-15 ◽

Cited By ~ 10

Author(s):

Dool-Ri Oh ◽

Yujin Kim ◽

Eun-jin Choi ◽

Hunmi-Lee ◽

Myung-A Jung ◽

...

Keyword(s):

Body Weight ◽

Adipose Tissue ◽

Bioactive Compounds ◽

Lipid Accumulation ◽

Weight Control ◽

Underlying Mechanism ◽

Serum Glucose ◽

Mrna Levels ◽

Dependent Manner

Background. The objective of the present study was to perform a bioguided fractionation of unripeRubus coreanusMiquel (uRC) and evaluate the lipid accumulation system involvement in its antiobesity activity as well as study the uRC mechanism of action.Results. After the fractionation, the BuOH fraction of uRC (uRCB) was the most active fraction, suppressing the differentiation of 3T3-L1 adipocytes in a dose-dependent manner. Moreover, after an oral administration for 8 weeks in HFD-induced obese mice, uRCB (10 and 50 mg/kg/day) produced a significant decrease in body weight, food efficiency ratio, adipose tissue weight and LDL-cholesterol, serum glucose, TC, and TG levels. Similarly, uRCB significantly suppressed the elevated mRNA levels of PPARγin the adipose tissuein vivo. Next, we investigated the antiobesity effects of ellagic acid, erycibelline, 5-hydroxy-2-pyridinemethanol, m-hydroxyphenylglycine, and 4-hydroxycoumarin isolated from uRCB. Without affecting cell viability, five bioactive compounds decreased the lipid accumulation in the 3T3-L1 cells and the mRNA expression levels of key adipogenic genes such as PPARγ, C/EBPα, SREBP-1c, ACC, and FAS.Conclusion. These results suggest that uRC and its five bioactive compounds may be a useful therapeutic agent for body weight control by downregulating adipogenesis and lipogenesis.

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Interferon-gamma regulation of Clara cell gene expression: in vivo and in vitro

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1997.272.6.l1142 ◽

1997 ◽

Vol 272 (6) ◽

pp. L1142-L1151 ◽

Cited By ~ 16

Author(s):

S. M. Magdaleno ◽

G. Wang ◽

K. J. Jackson ◽

M. K. Ray ◽

S. Welty ◽

...

Keyword(s):

Interferon Gamma ◽

Potential Interaction ◽

T Antigen ◽

Clara Cell ◽

Mrna Levels ◽

Dependent Manner ◽

Sv40 Large T Antigen ◽

Ifn Gamma

This report demonstrates that Clara cell 10-kDa protein (CC10) mRNA levels are regulated by interferon-gamma (IFN-gamma). An analysis of total lung RNA from mice given IFN-gamma intratracheally showed increased levels of CC10 mRNA compared to control animals but no significant increases in surfactant proteins B and C. These results were confirmed in a Clara cell line, mtCC1-2, generated from the lungs of a transgenic mouse expressing the SV40 large T antigen under the control of a Clara cell-specific promoter. Significant increases in mtCC1-2 CC10 mRNA levels were observed in a time- and a dose-dependent manner. The expression of transacting factors hepatocyte nuclear factors 3 alpha and 3 beta (HNF-3 alpha and HNF-3 beta) were also analyzed, and a transient increase in the expression of HNF-3 beta but not HNF-3 alpha was detected. Deoxyribonuclease I footprint analysis identified a signal transducer and activator of transcription (STAT) binding site (at nucleotides -293 to -284 of CC10) adjacent to two thyroid transcription factor-1 (TTF-1) binding sites, suggesting a potential interaction between STAT1 and TTF-1. This report reinforces the hypothesis that CC10 functions as an anti-inflammatory protein and that increases in CC10 protein may provide additional protection from inflammation and disease in the lung.

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Propionyl-L-carnitine Reduces Proliferation and Potentiates Bax-related Apoptosis of Aortic Intimal Smooth Muscle Cells by Modulating Nuclear Factor-κB Activity

Journal of Biological Chemistry ◽

10.1074/jbc.m606148200 ◽

2006 ◽

Vol 282 (7) ◽

pp. 4932-4942 ◽

Cited By ~ 25

Author(s):

Augusto Orlandi ◽

Arianna Francesconi ◽

Marcella Marcellini ◽

Antonio Di Lascio ◽

Luigi Giusto Spagnoli

Keyword(s):

Smooth Muscle ◽

Smooth Muscle Cells ◽

Cell Apoptosis ◽

Muscle Cells ◽

Nuclear Factor Κb ◽

Arterial Disease ◽

Antisense Oligodeoxynucleotide ◽

Dependent Manner ◽

Peripheral Arterial

Propionyl-l-carnitine (PLC) has been introduced among the therapeutic approaches of peripheral arterial disease, and more recently, an increase of intimal cell apoptosis has been demonstrated to contribute to its effectiveness in rabbit carotid postinjury myointimal hyperplasia prevention. How PLC mediates these effects on vascular smooth muscle cells (SMCs) remains poorly understood. We investigated the role of NF-κB in PLC-induced arterial remodeling. In vivo, daily PLC treatment 15 days after injury resulted in a reduction of relative rat aortic intimal volume, an increase of apoptosis, Bax up-regulation without changing the Bcl-2 level, and a reduction of NF-κB, vascular cell adhesion molecule-1, monocyte chemotactic protein-1, and survivin in myointimal thickening compared with controls. In the presence of 10% serum, a reduced G1 → S phase progression preceded PLC-induced intimal cell apoptosis; in 0.1% serum cultures, in a dose-dependent manner, PLC rapidly induced intimal cell apoptosis and reduced p65, p50, IAP-1, and IAP-2 expression. Inhibiting NF-κB activation through SN50 increased apoptotic rate and Bax expression in intimal but not in medial SMCs, and successive PLC treatment failed to induce a further increase in apoptotic rate. Bax antisense oligodeoxynucleotide reduced PLC-induced intimal cell apoptosis and cytochrome c release. The PLC-induced attenuation of NF-κB activity in intimal cells was also due to the increase of IκB-α bioavailability, as the result of a parallel induction of IκB-α synthesis and reduction of phosphorylation and degradation. Collectively, these findings document that NF-κB activity inhibition contributes to PLC-induced proliferative arrest and Bax-related apoptosis of intimal SMCs.

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