scholarly journals miR-27b promotes type II collagen expression by targetting peroxisome proliferator-activated receptor-γ2 during rat articular chondrocyte differentiation

2018 ◽  
Vol 38 (1) ◽  
Author(s):  
Jinying Xu ◽  
Shuang Lv ◽  
Yi Hou ◽  
Kan Xu ◽  
Dongjie Sun ◽  
...  
Keyword(s):  
Articular Cartilage ◽  
Type Ii Collagen ◽  
Luciferase Reporter ◽  
Peroxisome Proliferator ◽  
Dependent Manner ◽  
Type Ii ◽  
Peroxisome Proliferator Activated Receptor ◽  
Protein Levels ◽  
Pparγ Agonist ◽  
Gene Assay

MicroRNAs (miRNAs) play an essential role in articular cartilage development and growth. However, the exact mechanisms involved in this process remain unknown. In the present study, we investigated the biological functions of miR-27b during hypertrophic differentiation of rat articular chondrocytes. Based on in situ hybridization and immunohistochemistry, we report that miR-27b expression is reduced in the hypertrophic zone of articular cartilage, but expression of peroxisome proliferator-activated receptor γ (Pparγ) is increased. Dual-luciferase reporter gene assay and Western blot analysis demonstrated that Pparγ2 is a target of miR-27b. Overexpression of miR-27b inhibited expression of Pparγ2, as well as type X collagen (Col10a1) and matrix metalloproteinase 13 (Mmp13), while significantly promoting the expression of Sex-determining Region-box 9 (Sox9) and type II collagen (Col2a1) at both the mRNA and protein levels. Rosiglitazone, a Pparγ agonist, suppressed Col2a1 expression, while promoting expression of runt-related transcription factor 2 (Runx2) and Col10a1 in a concentration-dependent manner. siRNA-mediated knockdown of Pparγ2 caused an increase in protein levels of Col2a1. The present study demonstrates that miR-27b regulates chondrocyte hypertrophy in part by targetting Pparγ2, and that miR-27b may have important therapeutic implications in cartilage diseases.

scholarly journals Comprehensive analysis of PPARγ agonist activities of stereo-, regio-, and enantio-isomers of hydroxyoctadecadienoic acids

2020 ◽  
Vol 40 (4) ◽  
Author(s):  
Aya Umeno ◽  
Mami Sakashita ◽  
Sakiko Sugino ◽  
Kazutoshi Murotomi ◽  
Tsugumi Okuzawa ◽  
...  
Keyword(s):  
Early Stage ◽  
Biological Activities ◽  
Docking Simulation ◽  
Luciferase Reporter ◽  
Water Ligand ◽  
Peroxisome Proliferator ◽  
Agonist Activity ◽  
Peroxisome Proliferator Activated Receptor ◽  
Pparγ Agonist

Abstract Hydroxyoctadecadienoic acids (HODEs) are produced by oxidation and reduction of linoleates. There are several regio- and stereo-isomers of HODE, and their concentrations in vivo are higher than those of other lipids. Although conformational isomers may have different biological activities, comparative analysis of intracellular function of HODE isomers has not yet been performed. We evaluated the transcriptional activity of peroxisome proliferator-activated receptor γ (PPARγ), a therapeutic target for diabetes, and analyzed PPARγ agonist activity of HODE isomers. The lowest scores for docking poses of 12 types of HODE isomers (9-, 10-, 12-, and 13-HODEs) were almost similar in docking simulation of HODEs into PPARγ ligand-binding domain (LBD). Direct binding of HODE isomers to PPARγ LBD was determined by water-ligand observed via gradient spectroscopy (WaterLOGSY) NMR experiments. In contrast, there were differences in PPARγ agonist activities among 9- and 13-HODE stereo-isomers and 12- and 13-HODE enantio-isomers in a dual-luciferase reporter assay. Interestingly, the activity of 9-HODEs was less than that of other regio-isomers, and 9-(E,E)-HODE tended to decrease PPARγ-target gene expression during the maturation of 3T3-L1 cells. In addition, 10- and 12-(Z,E)-HODEs, which we previously proposed as biomarkers for early-stage diabetes, exerted PPARγ agonist activity. These results indicate that all HODE isomers have PPARγ-binding affinity; however, they have different PPARγ agonist activity. Our findings may help to understand the biological function of lipid peroxidation products.

Abstract 1729: PPARγ Regulates ABCG1 Expression in Macrophages via LXR-Driven Dual Promoters

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Makoto Ayaori ◽  
Masatsune Ogura ◽  
Kazuhiro Nakaya ◽  
Tetsuya Hisada ◽  
Shun-ichi Takiguchi ◽  
...  
Keyword(s):  
Cholesterol Efflux ◽  
Density Lipoprotein ◽  
Peroxisome Proliferator ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Peroxisome Proliferator Activated Receptor ◽  
Protein Levels ◽  
Exon 1 ◽  
Dual Promoters ◽  
Sirna Knockdown

ATP binding cassette transporter G1 (ABCG1), which is expressed in macrophages, has been implicated in the efflux of cholesterol to high density lipoprotein. Peroxisome proliferator-activated receptor γ (PPARγ) has been reported to be involved in cholesterol efflux from macrophages, and increased expression of ABCG1 via liver receptor X (LXR)-dependent and independent pathways. However, the mechanisms by which ABCG1 expression is increased by PPARγ have not been fully characterized. We observed that pioglitazone, a PPARγ ligand, increases cholesterol efflux from THP-1 macrophages, as well as ABCG1 mRNA and protein levels. Treatment with actinomycin D abolished the inducible effect of pioglitazone on ABCG1, indicating that pioglitazone transcriptionally activated ABCG1 expression. To clarify how pioglitazone regulates ABCG1 expression, we investigated promoter activity using reporter constructs containing human ABCG1 promoter A and B (located upstream of exon 1 and 5, respectively), with or without mutated LXR-binding sites. The results indicated that pioglitazone activated both promoters in an LXR-dependent manner. We also observed that pioglitazone increased two major transcripts driven by promoter A and B using specific primers for each transcript. To determine whether PPARγ and LXRα were involved in these effects of pioglitazone, we performed siRNA-knockdown of PPARγ and LXRα in macrophages, which resulted in 75% and 91% decreases in PPARγ and LXRα mRNA levels, respectively. PPARγ and LXRα-knockdown, respectively, completely or partially abolished pioglitazone-induced ABCG1 expression. In conclusion, these results suggest that pioglitazone transcriptionally increased ABCG1 expression in macrophages by activating dual promoters in an LXR-dependent manner. Further studies are needed to assess LXR-independent mechanisms for the stimulatory effect of pioglitazone on ABCG1.

scholarly journals Peroxisome-Proliferator Receptor γ Represses Hepatic Sex Hormone-Binding Globulin Expression

Endocrinology ◽  
2009 ◽  
Vol 150 (5) ◽  
pp. 2183-2189 ◽  
Author(s):  
David M. Selva ◽  
Geoffrey L. Hammond
Keyword(s):  
Hepg2 Cells ◽  
Promoter Activity ◽  
Binding Activity ◽  
Sex Hormone Binding Globulin ◽  
Luciferase Reporter ◽  
Peroxisome Proliferator ◽  
Luciferase Reporter Gene ◽  
Pparγ Agonist ◽  
Gene Assay

Plasma SHBG production by the liver is influenced by its metabolic state, and hepatocyte nuclear factor-4α regulates SHBG expression in response to changes in lipogenesis. Peroxisome-proliferator receptors (PPARs) also regulate glucose homeostasis and fatty acid metabolism. The human SHBG promoter contains a PPAR-response element (PPAR-RE), and plasma SHBG levels increase in polycystic ovarian syndrome patients treated with the PPARγ agonist, rosiglitazone. In addition, plasma SHBG levels are associated with a genetic polymorphism in the PPARγ-2 coding sequence that alters its transcriptional activity. Therefore, we set out to determine whether PPARγ influences hepatic production of SHBG by using human HepG2 hepatoblastoma cells as an in vitro model. Surprisingly, treatment of HepG2 cells with rosiglitazone reduced SHBG production and SHBG promoter activity (as assessed in a luciferase reporter gene assay) by 20–25%, whereas the PPARγ antagonist, GW9662, increased both by 2- to 3-fold. The effects of PPARγ agonists and antagonists on SHBG promoter activity were substantially diminished when the PPAR-RE in the SHBG promoter was mutated. A PPARγ small interfering RNA also increased SHBG production by HepG2 cells as well as SHBG promoter activity, and the latter was accentuated by cotreatment with GW9662. Importantly, overexpression of a PPARγ-2 Pro12 variant in HepG2 cells was more effective at reducing SHBG promoter activity, when compared with PPARγ-2 Ala12, consistent with its superior PPAR-RE binding activity. We conclude that PPARγ represses human SHBG expression in liver cells, and that differences in PPARγ levels and activity contribute directly to variations in plasma SHBG levels.

Molecular mechanism of 1,25-dihydroxyvitamin D3 inhibition of adipogenesis in 3T3-L1 cells

2006 ◽  
Vol 290 (5) ◽  
pp. E916-E924 ◽  
Author(s):  
Juan Kong ◽  
Yan Chun Li
Keyword(s):  
Cell Differentiation ◽  
Molecular Mechanism ◽  
Regulatory Element ◽  
Peroxisome Proliferator ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Dihydroxyvitamin D3 ◽  
Peroxisome Proliferator Activated Receptor ◽  
Protein Levels

We have investigated the molecular mechanism whereby 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] inhibits adipogenesis in vitro. 1,25(OH)2D3 blocks 3T3-L1 cell differentiation into adipocytes in a dose-dependent manner; however, the inhibition is ineffective 24–48 h after the differentiation is initiated, suggesting that 1,25(OH)2D3 inhibits only the early events of the adipogenic program. Treatment of 3T3-L1 cells with 1,25(OH)2D3 does not block the mitotic clonal expansion or C/EBPβ induction; rather, 1,25(OH)2D3 blocks the expression of C/EBPα, peroxisome proliferator-activated receptor-γ (PPARγ), sterol regulatory element-binding protein-1, and other downstream adipocyte markers. The inhibition by 1,25(OH)2D3 is reversible, since removal of 1,25(OH)2D3 from the medium restores the adipogenic process with only a temporal delay. Interestingly, although the vitamin D receptor (VDR) protein is barely detectable in 3T3-L1 preadipocytes, its levels are dramatically increased during the early phase of adipogenesis, peaking at 4–8 h and subsiding afterward throughout the rest of the differentiation program; 1,25(OH)2D3 treatment appears to stabilize the VDR protein levels. Consistently, adenovirus-mediated overexpression of human (h) VDR in 3T3-L1 cells completely blocks the adipogenic program, confirming that VDR is inhibitory. Inhibition of adipocyte differentiation by 1,25(OH)2D3 is ameliorated by troglitazone, a specific PPARγ antagonist; conversely, hVDR partially suppresses the transacting activity of PPARγ but not of C/EBPβ or C/EBPα. Moreover, 1,25(OH)2D3 markedly suppresses C/EBPα and PPARγ mRNA levels in mouse epididymal fat tissue culture. Taken together, these data indicate that the blockade of 3T3-L1 cell differentiation by 1,25(OH)2D3 occurs at the postclonal expansion stages and involves direct suppression of C/EBPα and PPARγ upregulation, antagonization of PPARγ activity, and stabilization of the inhibitory VDR protein.

scholarly journals PGC-1α Signaling Increases GABA(A) Receptor Subunit α2 Expression, GABAergic Neurotransmission and Anxiety-Like Behavior in Mice

2021 ◽  
Vol 14 ◽  
Author(s):  
Taavi Vanaveski ◽  
Svetlana Molchanova ◽  
Dan Duc Pham ◽  
Annika Schäfer ◽  
Ceren Pajanoja ◽  
...  
Keyword(s):  
Peroxisome Proliferator ◽  
Peroxisome Proliferator Activated Receptor ◽  
Gabaergic Neurotransmission ◽  
Gaba A ◽  
Protein Levels ◽  
Ca1 Region ◽  
Hippocampal Ca1 Region ◽  
Hippocampal Ca1 ◽  
Pparγ Agonist

Peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) is a master regulator of mitochondria biogenesis and cell stress playing a role in metabolic and degenerative diseases. In the brain PGC-1α expression has been localized mainly to GABAergic interneurons but its overall role is not fully understood. We observed here that the protein levels of γ-aminobutyric acid (GABA) type A receptor-α2 subunit (GABARα2) were increased in hippocampus and brain cortex in transgenic (Tg) mice overexpressing PGC-1α in neurons. Along with this, GABARα2 expression was enhanced in the hippocampus of the PGC-1α Tg mice, as shown by quantitative PCR. Double immunostaining revealed that GABARα2 co-localized with the synaptic protein gephyrin in higher amounts in the striatum radiatum layer of the hippocampal CA1 region in the Tg compared with Wt mice. Electrophysiology revealed that the frequency of spontaneous and miniature inhibitory postsynaptic currents (mIPSCs) was increased in the CA1 region in the Tg mice, indicative of an augmented GABAergic transmission. Behavioral tests revealed an increase for anxiety-like behavior in the PGC-1α Tg mice compared with controls. To study whether drugs acting on PPARγ can affect GABARα2, we employed pioglitazone that elevated GABARα2 expression in primary cultured neurons. Similar results were obtained using the specific PPARγ agonist, N-(2-benzoylphenyl)-O-[2-(methyl-2-pyridinylamino) ethyl]-L-tyrosine hydrate (GW1929). These results demonstrate that PGC-1α regulates GABARα2 subunits and GABAergic neurotransmission in the hippocampus with behavioral consequences. This indicates further that drugs like pioglitazone, widely used in the treatment of type 2 diabetes, can influence GABARα2 expression via the PPARγ/PGC-1α system.

scholarly journals Statins Activate Human PPAR Promoter and Increase PPAR mRNA Expression and Activation in HepG2 Cells

PPAR Research ◽  
2008 ◽  
Vol 2008 ◽  
pp. 1-11 ◽  
Author(s):  
Makoto Seo ◽  
Ikuo Inoue ◽  
Masaaki Ikeda ◽  
Takanari Nakano ◽  
Seiichiro Takahashi ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Hepg2 Cells ◽  
Promoter Activity ◽  
Transcriptional Activity ◽  
Statin Treatment ◽  
Expression Vectors ◽  
Peroxisome Proliferator ◽  
Dependent Manner ◽  
Peroxisome Proliferator Activated Receptor ◽  
Protein Levels

Statins increase peroxisome proliferator-activated receptor (PPAR) mRNA expression, but the mechanism of this increased PPAR production remains elusive. To examine the regulation of PPAR production, we examined the effect of 7 statins (atorvastatin, cerivastatin, fluvastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin) on human PPAR promoter activity, mRNA expression, nuclear protein levels, and transcriptional activity. The main results are as follows. (1) Majority of statins enhanced PPAR promoter activity in a dose-dependent manner in HepG2 cells transfected with the human PPAR promoter. This enhancement may be mediated by statin-induced HNF-4. (2) PPAR mRNA expression was increased by statin treatment. (3) The PPAR levels in nuclear fractions were increased by statin treatment. (4) Simvastatin, pravastatin, and cerivastatin markedly enhanced transcriptional activity in 293T cells cotransfected with acyl-coenzyme A oxidase promoter and PPAR/RXR expression vectors. In summary, these data demonstrate that PPAR production and activation are upregulated through the PPAR promoter activity by statin treatment.

scholarly journals Peroxisome Proliferator-Activated Receptor-γ Increases Adiponectin Secretion via Transcriptional Repression of Endoplasmic Reticulum Chaperone Protein ERp44

Endocrinology ◽  
2010 ◽  
Vol 151 (7) ◽  
pp. 3195-3203 ◽  
Author(s):  
Qinqiang Long ◽  
Ting Lei ◽  
Bin Feng ◽  
Changjun Yin ◽  
Dan Jin ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Transcriptional Repression ◽  
Energy Homeostasis ◽  
Luciferase Reporter ◽  
Peroxisome Proliferator ◽  
Response Element ◽  
Peroxisome Proliferator Activated Receptor ◽  
Ppar Γ ◽  
Pparγ Agonist ◽  
Peroxisome Proliferator Response Element

Adiponectin, an adipocyte-derived hormone, is a versatile player involved in the regulation of energy homeostasis, cardiovascular disease, and diabetes. Within adipocytes, adiponectin is retained in the lumen of the endoplasmic reticulum (ER) by binding to the thiol protein ER resident protein 44 kDa (ERp44), which is apparently regulated by the activation of nuclear receptor peroxisome proliferator-activated receptor (PPAR)-γ. However, the precise role of ERp44 in adiponectin secretion remains elusive. In the present study, we investigated the functional correlation between ERp44 and adiponectin in a pig model. The transcription of porcine ERp44 was regulated by PPARγ, which was consistent with the finding of putative peroxisome proliferator response element sites within ERp44 promoter. Using chromatin immunoprecipitation and luciferase reporter assays, we demonstrated that the transcription of porcine ERp44 is repressed through binding of PPARγ to a peroxisome proliferator response element site located between positions −981 and −1004 in its 5′-flanking region. In human embryonic kidney 293 cells stably transfected with cDNA encoding porcine adiponectin, the secretion of adiponectin was significantly up-regulated and the ERp44 mRNA was down-regulated observably, by either the treatment of PPARγ agonist rosiglitazone or the overexpression of PPARγ in these cells. Taken together, our results indicated that PPARγ is an essential regulatory factor for the transcriptional activity of ERp44, which in turn controls the secretion of adiponectin.

scholarly journals Down-regulation of microRNA-216b inhibits IL-1β-induced chondrocyte injury by up-regulation of Smad3

2017 ◽  
Vol 37 (2) ◽  
Author(s):  
Jiye He ◽  
Jiahong Zhang ◽  
Dongliang Wang
Keyword(s):  
Cell Proliferation ◽  
Type Ii Collagen ◽  
Joint Disease ◽  
Luciferase Reporter ◽  
Type Ii ◽  
Articular Chondrocyte ◽  
Down Regulation ◽  
Protein Levels ◽  
Normal Tissues ◽  
Smad3 Expression

Osteoarthritis (OA) is the most common type of joint disease, leading to a major cause of pain and disability. OA is characterized by the continuous degradation of articular cartilage, mainly resulting in an imbalance between synthesis and degradation of articular chondrocyte extracellular matrix (ECM). Aberrant miR-216b expression has been found in multiple cancers. However, the level of miR-216b in OA cartilage and its role in progression of this disease are still unknown. In the present study, the functional roles of miR-216b and its expression in OA tissues and interleukin-1β (IL-1β)-induced chondrocytes were examined. We found that the level of miR-216b was significantly higher and Smad3 expression was obviously lower in OA cartilage and IL-1β-induced chondrocytes than in normal tissues and cells. Furthermore, a bioinformatics analysis and luciferase reporter assay identified Smad3 as a direct target gene of miR-216b, and Smad3 expression was reduced by miR-216b overexpression at both the mRNA and protein levels. A functional analysis demonstrated that miR-216b down-regulation obviously alleviated the IL-1β-induced inhibition in cell proliferation, type II collagen, and aggrecan down-regulation and matrix metalloproteinase-13 (MMP-13) up-regulation, while miR-216b overexpression had the opposite effects. Knockdown of Smad3 by siRNA reversed the effects of the miR-216b inhibitor on cell proliferation, the expressions of type II collagen, aggrecan, and MMP-13. Our results suggested that miR-216b contributes to progression of OA by directly targeting Smad3, providing a potential therapeutic target for treatment of OA.

scholarly journals Human lung fibroblasts produce proresolving peroxisome proliferator-activated receptor-γ ligands in a cyclooxygenase-2-dependent manner

2016 ◽  
Vol 311 (5) ◽  
pp. L855-L867 ◽  
Author(s):  
Shannon H. Lacy ◽  
Collynn F. Woeller ◽  
Thomas H. Thatcher ◽  
Krishna Rao Maddipati ◽  
Kenneth V. Honn ◽  
...  
Keyword(s):  
Human Lung ◽  
Cyclooxygenase 2 ◽  
Lung Fibroblasts ◽  
Luciferase Reporter ◽  
Chronic Obstructive ◽  
Peroxisome Proliferator ◽  
Dependent Manner ◽  
Peroxisome Proliferator Activated Receptor ◽  
Human Lung Fibroblasts ◽  
Cox 2

Human lung fibroblasts (HLFs) act as innate immune sentinel cells that amplify the inflammatory response to injurious stimuli. Here, we use targeted lipidomics to explore the hypothesis that HLFs also play an active role in the resolution of inflammation. We detected cyclooxygenase-2 (COX-2)-dependent production of both proinflammatory and proresolving prostaglandins (PGs) in conditioned culture medium from HLFs treated with a proinflammatory stimulus, IL-1β. Among the proresolving PGs in the HLF lipidome were several known ligands for peroxisome proliferator-activated receptor-γ (PPARγ), a transcription factor whose activation in the lung yields potent anti-inflammatory, antifibrotic, and proresolving effects. Next, we used a cell-based luciferase reporter to confirm the ability of HLF supernatants to activate PPARγ, demonstrating, for the first time, that primary HLFs activated with proinflammatory IL-1β or cigarette smoke extract produce functional PPARγ ligands; this phenomenon is temporally regulated, COX-2- and lipocalin-type PGD synthase-dependent, and enhanced by arachidonic acid supplementation. Finally, we used luciferase reporter assays to show that several of the PGs in the lipidome of activated HLFs independently activate PPARγ and/or inhibit NFκB. These results indicate that HLFs, as immune sentinels, regulate both proinflammatory and proresolving responses to injurious stimuli. This novel endogenous resolution pathway represents a new therapeutic target for globally important inflammatory diseases such as chronic obstructive pulmonary disease.

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