Overexpression Of Dominant Negative Peroxisome Proliferator-Activated Receptor-g Causes Pulmonary Inflammation And Emphysema

PPARG, a ligand-activated transcription factor plays a critical role in the regulation of blood pressure and vascular function. We hypothesized that smooth muscle cell (SMC) PPARG protects against hypertension (HT) and resistance vessel dysfunction. Transgenic mice expressing dominant negative PPARG (S-P467L) in SMC or non-transgenic controls (NT) were implanted with DOCA pellet and allowed ad libitum access to 0.15 M NaCl for 21 days in addition to regular chow and water. Blood pressure was monitored by telemetry and mesenteric arterial (MA) function was assessed by pressurized myograph. At baseline, 24-hour mean arterial pressure (MAP) was similar between NT and S-P467L mice, while the transgenic mice were tachycardic. DOCA-salt increased MAP to a much greater degree in S-P467L mice (Δ MAP; S-P467L: +34.2±6.0, NT: +13.3±5.7, p<0.05 vs NT). Heart rate was similarly decreased in both groups after DOCA-salt. Vasoconstriction to KCl, phenylephrine and endothelin-1 did not differ in MA from DOCA-salt treated NT and S-P467L, while the response to vasopressin was significantly reduced in S-P467L after DOCA-salt (% constriction at 10-8 M, S-P467L: 31.6±5.6, NT: 46.7±3.8, p<0.05 vs NT). Urinary copeptin, a surrogate marker for arginine vasopressin was similar in both groups regardless of treatment. Vasorelaxation to acetylcholine was slightly impaired in S-P467L MA compared to NT at baseline whereas this effect was further exaggerated after DOCA-salt (% relaxation at 10-5 M, S-P467L: 56.1±8.3, NT: 79.4±5.6, p<0.05 vs NT). Vascular morphology at luminal pressure of 75 mmHg showed a significant increase in wall thickness (S-P467L: 18.7±0.8, NT: 16.0±0.4, p<0.05 vs NT) and % media/lumen (S-P467L: 8.4±0.3, NT: 7.1±0.2, p<0.05 vs NT) in S-P467L MA after DOCA-salt. Expression of tissue inhibitor of metalloproteinases (TIMP)-4 and regulator of G-protein signaling (RGS)-5 transcript were 2- and 3.5-fold increased, respectively, in MA of NT with DOCA-salt compared to NT baseline. However, this induction was markedly blunted in S-P467L MA. We conclude that interference with PPARG function in SMC leads to altered gene expression crucial for normal vascular homeostasis, thereby sensitizing the mice to the effects of DOCA-salt induced HT and vascular dysfunction.

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PPARγ regulates the function of human dendritic cells primarily by altering lipid metabolism

Blood ◽

10.1182/blood-2007-06-096222 ◽

2007 ◽

Vol 110 (9) ◽

pp. 3271-3280 ◽

Cited By ~ 81

Author(s):

Istvan Szatmari ◽

Daniel Töröcsik ◽

Maura Agostini ◽

Tibor Nagy ◽

Mark Gurnell ◽

...

Keyword(s):

Dendritic Cells ◽

Lipid Metabolism ◽

Receptor Activation ◽

Dominant Negative ◽

Transcriptional Level ◽

Peroxisome Proliferator ◽

Peroxisome Proliferator Activated Receptor ◽

Positive Effects ◽

Dominant Negative Mutation ◽

Human Dendritic Cells

Abstract Activation of the lipid-regulated nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) modifies the immunophenotype of monocyte-derived dendritic cells (DCs). However it has not been analyzed in a systematic manner how lipid metabolism and immune regulation are connected at the transcriptional level via this receptor. Here we present the genome-wide expression analyses of PPARγ-instructed human DCs. Receptor activation was achieved by exogenous, synthetic as well as endogenous, natural means. More than 1000 transcripts are regulated during DC development by activation of PPARγ; half of the changes are positive effects. These changes appear to enhance and modulate the robust gene expression alterations associated with monocyte to DC transition. Strikingly, only genes related to lipid metabolism are overrepresented among early induced genes. As a net consequence, lipid accumulation appears to be diminished in these cells. In contrast, genes related to immune response are regulated after 24 hours, implying the existence of indirect mechanisms of modulation. Receptor dependence was established by using DCs of patients harboring a dominant-negative mutation of PPARγ. Our data show that PPARγ acts as a mostly positive transcriptional regulator in human developing DCs, acting primarily through controlling genes involved in lipid metabolism and via this, indirectly modifying the immune phenotype.

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CREB Activation Induces Adipogenesis in 3T3-L1 Cells

Molecular and Cellular Biology ◽

10.1128/mcb.20.3.1008-1020.2000 ◽

2000 ◽

Vol 20 (3) ◽

pp. 1008-1020 ◽

Cited By ~ 213

Author(s):

Jane E. B. Reusch ◽

Lilliester A. Colton ◽

Dwight J. Klemm

Keyword(s):

Transcription Factor ◽

Dominant Negative ◽

Marker Genes ◽

Peroxisome Proliferator ◽

Peroxisome Proliferator Activated Receptor ◽

Fatty Acid Binding ◽

Fat Composition ◽

Specific Factors ◽

Necessary And Sufficient ◽

Physiological And Biochemical

ABSTRACT Obesity is the result of numerous, interacting behavioral, physiological, and biochemical factors. One increasingly important factor is the generation of additional fat cells, or adipocytes, in response to excess feeding and/or large increases in body fat composition. The generation of new adipocytes is controlled by several “adipocyte-specific” transcription factors that regulate preadipocyte proliferation and adipogenesis. Generally these adipocyte-specific factors are expressed only following the induction of adipogenesis. The transcription factor(s) that are involved in initiating adipocyte differentiation have not been identified. Here we demonstrate that the transcription factor, CREB, is constitutively expressed in preadipocytes and throughout the differentiation process and that CREB is stimulated by conventional differentiation-inducing agents such as insulin, dexamethasone, and dibutyryl cAMP. Stably transfected 3T3-L1 preadipocytes were generated in which we could induce the expression of either a constitutively active CREB (VP16-CREB) or a dominant-negative CREB (KCREB). Inducible expression of VP16-CREB alone was sufficient to initiate adipogenesis as determined by triacylglycerol storage, cell morphology, and the expression of two adipocyte marker genes, peroxisome proliferator activated receptor gamma 2, and fatty acid binding protein. Alternatively, KCREB alone blocked adipogenesis in cells treated with conventional differentiation-inducing agents. These data indicate that activation of CREB was necessary and sufficient to induce adipogenesis. Finally, CREB was shown to bind to putative CRE sequences in the promoters of several adipocyte-specific genes. These data firmly establish CREB as a primary regulator of adipogenesis and suggest that CREB may play similar roles in other cells and tissues.

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LKB1 signalling attenuates early events of adipogenesis and responds to adipogenic cues

Journal of Molecular Endocrinology ◽

10.1530/jme-13-0296 ◽

2014 ◽

Vol 53 (1) ◽

pp. 117-130 ◽

Cited By ~ 12

Author(s):

Amélie Gormand ◽

Christine Berggreen ◽

Lahouari Amar ◽

Emma Henriksson ◽

Ingrid Lund ◽

...

Keyword(s):

Transcription Factors ◽

Histone Deacetylases ◽

Fatty Acid Synthase ◽

Nuclear Translocation ◽

Adipogenic Differentiation ◽

Camp Response Element Binding ◽

Dominant Negative ◽

Hormone Sensitive Lipase ◽

Peroxisome Proliferator ◽

Peroxisome Proliferator Activated Receptor

cAMP-response element-binding protein (CREB) is required for the induction of adipogenic transcription factors such as CCAAT/enhancer-binding proteins (C/EBPs). Interestingly, it is known from studies in other tissues that LKB1 and its substrates AMP-activated protein kinase (AMPK) and salt-inducible kinases (SIKs) negatively regulate gene expression by phosphorylating the CREB co-activator CRTC2 and class IIa histone deacetylases (HDACs), which results in their exclusion from the nucleus where they co-activate or inhibit their targets. In this study, we show that AMPK/SIK signalling is acutely attenuated during adipogenic differentiation of 3T3-L1 preadipocytes, which coincides with the dephosphorylation and nuclear translocation of CRTC2 and HDAC4. When subjected to differentiation, 3T3-L1 preadipocytes in which the expression of LKB1 was stably reduced using shRNA (Lkb1-shRNA), as well as Lkb1-knockout mouse embryonic fibroblasts (Lkb1−/− MEFs), differentiated more readily into adipocyte-like cells and accumulated more triglycerides compared with scrambled-shRNA-expressing 3T3-L1 cells or Wt MEFs. In addition, the phosphorylation of CRTC2 and HDAC4 was reduced, and the mRNA expression of adipogenic transcription factors Cebpa, peroxisome proliferator-activated receptor γ (Pparg) and adipocyte-specific proteins such as hormone-sensitive lipase (HSL), fatty acid synthase (FAS), aP2, GLUT4 and adiponectin was increased in the absence of LKB1. The mRNA and protein expression of Ddit3/CHOP10, a dominant-negative member of the C/EBP family, was reduced in Lkb1-shRNA-expressing cells, providing a potential mechanism for the up-regulation of Pparg and Cebpa expression. These results support the hypothesis that LKB1 signalling keeps preadipocytes in their non-differentiated form.

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Examination of Ligand-Dependent Coactivator Recruitment by Peroxisome Proliferator-Activated Receptor-α(PPARα)

PPAR Research ◽

10.1155/ppar/2006/69612 ◽

2006 ◽

Vol 2006 ◽

pp. 1-9 ◽

Cited By ~ 11

Author(s):

Eric S. Tien ◽

Daniel B. Hannon ◽

Jerry T. Thompson ◽

John P. Vanden Heuvel

Keyword(s):

Conformational Change ◽

Peptide Library ◽

Dominant Negative ◽

Carboxyl Terminus ◽

Peroxisome Proliferator ◽

Agonist Activity ◽

Peroxisome Proliferator Activated Receptor ◽

Steroid Receptor Coactivator ◽

Important Protein ◽

Receptor Modulators

The ligand-dependent recruitment of coactivators to peroxisome proliferator-activated receptor-α(PPARα) was examined. PPAR-binding protein (PBP), PPARγcoactivator-1α(PGC-1α), steroid receptor coactivator-1 (SRC-1), and CBP/p300-interacting transactivator with ED-rich tail 2 (CITED2) affected PPARαactivity in the presence of Wy-14,643. The effects on PPARαactivity in light of increased or decreased expression of these coactivators were qualitatively different depending on the ligand examined. Diminished expression of PGC-1α, SRC-1, or PBP by RNAi plasmids affected natural or synthetic agonist activity whereas only Wy-14,643 was affected by decreased PGC-1α. The interaction of PPARαwith an LXXLL-containing peptide library showed ligand-specific patterns, indicative of differences in conformational change. The association of coactivators to PPARαoccurs predominantly via the carboxyl-terminus and mutating456LHPLL to456LHPAA resulted in a dominant-negative construct. This research confirms that coactivator recruitment to PPARαis ligand-dependent and that selective receptor modulators (SRMs) of this important protein are likely.

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Impaired Peroxisome Proliferator-Activated Receptor γ Function through Mutation of a Conserved Salt Bridge (R425C) in Familial Partial Lipodystrophy

Molecular Endocrinology ◽

10.1210/me.2006-0485 ◽

2007 ◽

Vol 21 (5) ◽

pp. 1049-1065 ◽

Cited By ~ 28

Author(s):

Ellen H. Jeninga ◽

Olivier van Beekum ◽

Aalt D. J. van Dijk ◽

Nicole Hamers ◽

Brenda I. Hendriks-Stegeman ◽

...

Keyword(s):

Retinoic Acid ◽

Transcriptional Activity ◽

Salt Bridge ◽

Dominant Negative ◽

Heterozygous Mutation ◽

Peroxisome Proliferator ◽

Wild Type ◽

Peroxisome Proliferator Activated Receptor ◽

Partial Lipodystrophy ◽

Familial Partial Lipodystrophy

Abstract The nuclear receptor peroxisome proliferator-activated receptor (PPAR) γ plays a key role in the regulation of glucose and lipid metabolism in adipocytes by regulating their differentiation, maintenance, and function. A heterozygous mutation in the PPARG gene, which changes an arginine residue at position 425 into a cysteine (R425C), has been reported in a patient with familial partial lipodystrophy subtype 3 (FPLD3). The strong conservation of arginine 425 among nuclear receptors that heterodimerize with retinoic acid X receptor prompted us to investigate the functional consequences of the R425C mutation on PPARγ function. Here we show that this mutant displayed strongly reduced transcriptional activity compared with wild-type PPARγ, irrespective of cell type, promoter context, or ligand, whereas transrepression of nuclear factor-κB activity remained largely intact. Our data indicate that the reduced transcriptional activity of PPARγ R425C is not caused by impaired corepressor release, but due to reduced dimerization with retinoic acid X receptor α in combination with reduced ligand binding and subsequent coactivator binding. As a consequence of these molecular defects, the R425C mutant was less effective in inducing adipocyte differentiation. PPARγ R425C did not inhibit its wild-type counterpart in a dominant-negative manner, suggesting a haploinsufficiency mechanism in at least some FPLD3 patients. Using molecular dynamics simulations, substitution of R425 with cysteine is predicted to cause the formation of an alternative salt bridge. This structural change provides a likely explanation of how mutation of a single conserved residue in a patient with FPLD3 can disrupt the function of the adipogenic transcription factor PPARγ on multiple levels.

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Peroxisome Proliferator-Activated Receptor γ Recruits the Positive Transcription Elongation Factor b Complex to Activate Transcription and Promote Adipogenesis

Molecular Endocrinology ◽

10.1210/me.2005-0222 ◽

2006 ◽

Vol 20 (7) ◽

pp. 1494-1505 ◽

Cited By ~ 67

Author(s):

Irena Iankova ◽

Rasmus K. Petersen ◽

Jean-Sébastien Annicotte ◽

Carine Chavey ◽

Jacob B. Hansen ◽

...

Keyword(s):

Transcription Elongation ◽

Elongation Factor ◽

Dominant Negative ◽

Peroxisome Proliferator ◽

Peroxisome Proliferator Activated Receptor ◽

Factor B ◽

Cyclin T1 ◽

Positive Effects ◽

Positive Transcription Elongation Factor ◽

Transcription Elongation Factor

Abstract Positive transcription elongation factor b (P-TEFb) phosphorylates the C-terminal domain of RNA polymerase II, facilitating transcriptional elongation. In addition to its participation in general transcription, P-TEFb is recruited to specific promoters by some transcription factors such as c-Myc or MyoD. The P-TEFb complex is composed of a cyclin-dependent kinase (cdk9) subunit and a regulatory partner (cyclin T1, cyclin T2, or cyclin K). Because cdk9 has been shown to participate in differentiation processes, such as muscle cell differentiation, we studied a possible role of cdk9 in adipogenesis. In this study we show that the expression of the cdk9 p55 isoform is highly regulated during 3T3-L1 adipocyte differentiation at RNA and protein levels. Furthermore, cdk9, as well as cyclin T1 and cyclin T2, shows differences in nuclear localization at distinct stages of adipogenesis. Overexpression of cdk9 increases the adipogenic potential of 3T3-L1 cells, whereas inhibition of cdk9 by specific cdk inhibitors, and dominant-negative cdk9 mutant impairs adipogenesis. We show that the positive effects of cdk9 on the differentiation of 3T3-L1 cells are mediated by a direct interaction with and phosphorylation of peroxisome proliferator-activated receptor γ (PPARγ), which is the master regulator of this process, on the promoter of PPARγ target genes. PPARγ-cdk9 interaction results in increased transcriptional activity of PPARγ and therefore increased adipogenesis.

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Inhibition of PPARγ in myeloid-lineage cells induces systemic inflammation, immunosuppression, and tumorigenesis

Blood ◽

10.1182/blood-2011-06-363093 ◽

2012 ◽

Vol 119 (1) ◽

pp. 115-126 ◽

Cited By ~ 53

Author(s):

Lingyan Wu ◽

Cong Yan ◽

Magdalena Czader ◽

Oded Foreman ◽

Janice S. Blum ◽

...

Keyword(s):

T Cells ◽

Cd4 T Cells ◽

Suppressor Cells ◽

Dominant Negative ◽

Peroxisome Proliferator ◽

Myeloid Lineage ◽

Peroxisome Proliferator Activated Receptor ◽

Anti Inflammatory ◽

Spleen And Lymph Nodes

Abstract Peroxisome proliferator–activated receptor-γ (PPARγ) is an anti-inflammatory molecule. To study its biologic function in myeloid cells, dominant-negative PPARγ (dnPPARγ) was overexpressed in a myeloid-specific bitransgenic mouse model. In this bitransgenic system, overexpression of the dnPPARγ-Flag fusion protein in myeloid-lineage cells abnormally elevated frequencies and total numbers of IL-7Rα−Lin−c-Kit+Sca-1−, Lin−/Scal+/c-Kit+, common myeloid, and granulocyte-monocyte progenitor populations in the BM. dnPPARγ overexpression led to up-regulation of IL-1β, IL-6, and TNFα in the blood plasma. As a result, CD11b+Ly6G+ cells were systemically increased in association with activation of Stat3, NF-κB, Erk1/2, and p38 molecules. Myeloid-derived suppressor cells (MDSCs) inhibited the proliferation and lymphokine production of wild-type CD4+ T cells in vitro. CD4+ T cells from doxycycline-treated bitransgenic mice displayed reduced proliferation and lymphokine release. Both CD4+ and CD8+ T-cell populations were decreased in doxycycline-treated bitransgenic mice. Multiple forms of carcinoma and sarcoma in the lung, liver, spleen, and lymph nodes were observed in doxycycline-treated bitransgenic mice. BM transplantation revealed that a myeloid-autonomous defect was responsible for MDSC expansion, immunosuppression, and tumorigenesis in these mice. These studies suggest that anti-inflammatory PPARγ in myeloid-lineage cells plays a key role in controlling pro-inflammatory cytokine synthesis, MDSC expansion, immunosuppression, and the development of cancer.

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Abstract 10: Smooth Muscle Peroxisome Proliferator-Activated Receptor γ Plays a Critical Role in Formation and Rupture of Cerebral Aneurysms in Mice in vivo

Stroke ◽

10.1161/str.47.suppl_1.10 ◽

2016 ◽

Vol 47 (suppl_1) ◽

Author(s):

Robert M Starke ◽

He Gu ◽

Katina Wilson ◽

Yi Chu ◽

Nohra Chalouhi ◽

...

Keyword(s):

Smooth Muscle ◽

Vascular Inflammation ◽

Critical Role ◽

Cerebral Aneurysms ◽

Dominant Negative ◽

Peroxisome Proliferator ◽

Nuclear Factor ◽

Peroxisome Proliferator Activated Receptor ◽

Aneurysm Formation ◽

Pparγ Agonist

Background and Purpose: Vascular inflammation plays a critical role in the pathogenesis of cerebral aneurysms. Peroxisome proliferator-activated receptor γ (PPARγ) protects against vascular inflammation and atherosclerosis, whereas dominant-negative mutations in PPARγ promote atherosclerosis and vascular dysfunction. In this study, the role of PPARγ in aneurysm formation and rupture was tested. Methods: Aneurysms were induced with a combination of systemic infusion of angiotensin-II and local injection of elastase in (1) mice that received the PPARγ antagonist GW9662 or the PPARγ agonist pioglitazone, (2) mice carrying dominant-negative PPARγ mutations in endothelial or smooth muscle cells, and (3) mice that received the Cullin inhibitor MLN4924. Incidence of aneurysm formation, rupture, and mortality was quantified. Cerebral arteries were analyzed for expression of Cullin3, Kelch-like ECH-associated protein 1, nuclear factor (erythroid-derived 2)-like 2, NAD(P)H dehydrogenase (quinone)1 (NQO1), and inflammatory marker mRNAs. Results: Neither pioglitazone nor GW9662 altered the incidence of aneurysm formation. GW9662 significantly increased the incidence of aneurysm rupture, whereas pioglitazone tended to decrease the incidence of rupture. Dominant-negative endothelial-specific PPARγ did not alter the incidence of aneurysm formation or rupture. In contrast, dominant-negative smooth muscle-specific PPARγ resulted in an increase in aneurysm formation (P<0.05) and rupture (P=0.05). Dominant-negative smooth muscle-specific PPARγ, but not dominant-negative endothelial-specific PPARγ, resulted in significant decreases in expression of genes encoding Cullin3, Kelch-like ECH-associated protein 1, and nuclear factor (erythroid-derived 2)-like 2, along with significant increases in tumor necrosis factor-α, monocyte chemoattractant protein-1, chemokine (C-X-C motif) ligand 1, CD68, matrix metalloproteinase-3, -9, and -13. MLN4924 did not alter incidence of aneurysm formation, but increased the incidence of rupture (P<0.05). Conclusions: Endogenous PPARγ, specifically smooth muscle PPARγ, plays an important role in protecting from formation and rupture of experimental cerebral aneurysms in mice.

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