Emerging PPAR γ

Peroxisome proliferator activated receptor (PPAR)-γ is a nuclear hormone receptor that is activated by multiple agonists including thiazolidinediones, prostaglandins, and synthetic oleanolic acids. Many PPARγ ligands are under investigation as potential therapies for human diseases. These ligands modulate multiple cellular pathways via both PPARγ-dependent and PPARγ-independent mechanisms. Here, we review the role of PPARγ and PPARγ ligands in lung disease, with emphasis on PPARγ-independent effects. PPARγ ligands show great promise in moderating lung inflammation, as antiproliferative agents in combination to enhance standard chemotherapy in lung cancer and as treatments for pulmonary fibrosis, a progressive fatal disease with no effective therapy. Some of these effects occur when PPARγ is pharmaceutically antagonized or genetically PPARγ and are thus independent of classical PPARγ-dependent transcriptional control. Many PPARγ ligands demonstrate direct binding to transcription factors and other proteins, altering their function and contributing to PPARγ-independent inhibition of disease phenotypes. These PPARγ-independent mechanisms are of significant interest because they suggest new therapeutic uses for currently approved drugs and because they can be used as probes to identify novel proteins and pathways involved in the pathogenesis or treatment of disease, which can then be targeted for further investigation and drug development

TNF-α and NF-κB Signaling Play a Critical Role in Cigarette Smoke-induced Epithelial-mesenchymal Transition of Retinal Pigment Epithelial Cells in Proliferative Vitreoretinopathy

Proliferative vitreoretinopathy (PVR) is characterized by the growth and contraction of cellular membranes within the vitreous cavity and on both surfaces of the retina, resulting in recurrent retinal detachments and poor visual outcomes. Proinflammatory cytokines like tumor necrosis factor alpha (TNFα) have been associated with PVR and the epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells. Cigarette smoke is the only known modifiable risk factor for PVR, but the mechanisms are unclear. The purpose of this study was to examine the impact of cigarette smoke on the proinflammatory TNFα/NF-κB/Snail pathway in RPE cells to better understand the mechanisms through which cigarette smoke increases the risk of PVR. Human ARPE-19 cells were exposed to cigarette smoke extract (CSE), for 4 to 24-hours and TNFα, Snail, IL-6, IL-8, and α-SMA levels were analyzed by qPCR and/or Western blot. The severity of PVR formation was assessed in a murine model of PVR after intravitreal injection of ARPE-19 cells pre-treated with CSE or not. Fundus imaging, OCT imaging, and histologic analysis 4 weeks after injection were used to examine PVR severity. ARPE-19 cells exposed to CSE expressed higher levels of TNFα, SNAIL, IL6 and IL8 mRNA as well as SNAIL, Vimentin and α-SMA protein. Inhibition of TNFα and NF-κB pathways blocked the effect of CSE. In vivo, intravitreal injection of ARPE-19 cells treated with CSE resulted in more severe PVR compared to mice injected with untreated RPE cells. These studies suggest that the TNFα pathway is involved in the mechanism whereby cigarette smoke increases PVR. Further investigation into the role of TNFα/NF-κB/Snail in driving PVR and pharmacological targeting of these pathways in disease are warranted

NMD resulting from encephalomyocarditis virus IRES-directed translation initiation seems to be restricted to CBP80/20-bound mRNA

Nonsense-mediated messenger RNA decay (NMD) generally degrades mRNAs that prematurely terminate translation as a means of quality control. NMD in mammalian cells targets newly spliced mRNA that is bound by the cap-binding protein heterodimer CBP80/20 and one or more post-splicing exon junction complexes during a pioneer round of translation. NMD targets mRNA that initiates translation using the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES), therefore NMD might target not only CBP80/20-bound mRNA but also its remodelled product, eIF4E-bound mRNA. Here, we provide evidence that NMD triggered by translation initiation at the EMCV IRES, similar to NMD triggered by translation initiation at an mRNA cap, targets CBP80/20-bound mRNA but does not detectably target eIF4E-bound mRNA. We show that EMCV IRES-initiated translation undergoes a CBP80/20-associated pioneer round of translation that results in CBP80/20-dependent and Upf factor-dependent NMD when translation terminates prematurely

Microparticle composition influences inflammatory cytokine production from activated monocytes.

<p>THP-1 cells (<b>A</b>) or CD14 sorted blood monocytes (<b>B</b>) were treated with control microparticles (grey bars) or PPARγ-overexpressing microparticles (black bars) or no microparticles (white bars) for 4 hours before activation with LPS or PAM3CSK4 for 24 hours. Supernatants were collected and pro-inflammatory cytokines IL-8 (top), MCP-1 (bottom) were measured by ELISA. Dotted line represents baseline cytokine production from unactivated cells with no microparticle exposure. Mean values with standard errors represent one of at least 3 experiments. Two-way ANOVA with Tukey's multiple comparison post test was performed to determine statistical significance. * indicates (p<0.05).</p

Microparticles Engineered to Highly Express Peroxisome Proliferator-Activated Receptor-γ Decreased Inflammatory Mediator Production and Increased Adhesion of Recipient Monocytes

<div><p>Circulating blood microparticles are submicron vesicles released primarily by megakaryocytes and platelets that act as transcellular communicators. Inflammatory conditions exhibit elevated blood microparticle numbers compared to healthy conditions. Direct functional consequences of microparticle composition, especially internal composition, on recipient cells are poorly understood. Our objective was to evaluate if microparticle composition could impact the function of recipient cells, particularly during inflammatory provocation. We therefore engineered the composition of megakaryocyte culture-derived microparticles to generate distinct microparticle populations that were given to human monocytes to assay for influences recipient cell function. Herein, we tested the responses of monocytes exposed to either control microparticles or microparticles that contain the anti-inflammatory transcription factor, peroxisome proliferator-activated receptor-γ (PPARγ). In order to normalize relative microparticle abundance from two microparticle populations, we implemented a novel approach that utilizes a Nanodrop Spectrophotometer to assay for microparticle density rather than concentration. We found that when given to peripheral blood mononuclear cells, microparticles were preferentially internalized by CD11b+ cells, and furthermore, microparticle composition had a profound functional impact on recipient monocytes. Specifically, microparticles containing PPARγ reduced activated monocyte production of the proinflammatory cytokines interleukin-8 and monocyte chemotactic protein-1 compared to activated monocytes exposed to control microparticles. Additionally, treatment with PPARγ microparticles greatly increased monocyte cell adherence. This change in morphology occurred simultaneously with increased production of the key extracellular matrix protein, fibronectin and increased expression of the fibronectin-binding integrin, ITGA5. PPARγ microparticles also changed monocyte mRNA levels of several genes including those under PPARγ control. Overall, the delivery of PPARγ from microparticles to human monocytes influenced gene expression, decreased inflammatory mediator production and increased monocyte adherence. These results support the concept that the composition of blood microparticles has a profound impact on the function of cells with which they interact, and likely plays a role in vascular inflammation.</p></div

PPARγ-containing microparticles (PPARγ MP) increase adherence and spreading of THP-1 cells, but not lipid uptake.

<p>THP-1 cells were treated with microparticles for 4 hours and left unactivated or activated with LPS or PAM3CSK4 for 24 hours. Cells were removed from culture wells, which were then washed with phosphate buffered saline. <b>A</b>, Cells that were still attached to the wells were visualized with the 40× objective lens of an inverted microscope, and representative images from at least three independent experiments are shown. Of note, some of the PPARγ MP treated cells that were attached had a morphology indicative of spreading (arrows). <b>B</b>, Attached cells from five fields of view in each condition were quantified, and PPARγ MP treated cells always contained the most adherent cells. Two-way ANOVA with Tukey's multiple comparison post test was performed to determine statistical significance. Mean values with standard errors averaged from 5 experiments. * indicates (p<0.05).</p

Ogerin mediated inhibition of TGF-β(1) induced myofibroblast differentiation is potentiated by acidic pH.

Transforming growth factor beta (TGF-β) induced myofibroblast differentiation is central to the pathological scarring observed in Idiopathic Pulmonary Fibrosis (IPF) and other fibrotic diseases. Our lab has recently identified expression of GPR68 (Ovarian Cancer Gene Receptor 1, OGR1), a pH sensing G-protein coupled receptor, as a negative regulator of TGF-β induced profibrotic effects in primary human lung fibroblasts (PHLFs). We therefore hypothesized that small molecule activators of GPR68 would inhibit myofibroblast differentiation. Ogerin is a positive allosteric modulator (PAM) of GPR68, inducing a leftward shift of the dose response curve to proton induced signaling. Using PHLFs derived from patients with both non-fibrotic and IPF diagnoses, we show that Ogerin inhibits, and partially reverses TGF-β induced myofibroblast differentiation in a dose dependent manner. This occurs at the transcriptional level without inhibition of canonical TGF-β induced SMAD signaling. Ogerin induces PKA dependent CREB phosphorylation, a marker of Gαs pathway activation. The ability of Ogerin to inhibit both basal and TGF-β induced collagen gene transcription, and induction of Gαs signaling is enhanced at an acidic pH (pH 6.8). Similar findings were also found using fibroblasts derived from dermal, intestinal, and orbital tissue. The biological role of GPR68 in different tissues, cell types, and disease states is an evolving and emerging field. This work adds to the understanding of Gαs coupled GPCRs in fibrotic lung disease, the ability to harness the pH sensing properties of GPR68, and conserved mechanisms of fibrosis across different organ systems

PPARγ-containing microparticles (PPARγ MP) or direct transduction of PPARγ increase fibronectin production and ITGA5 expression of THP-1 cells.

<p><b>A–C</b>, THP-1 cells were treated with Control MP (grey bars), PPAR MP (black bars), or no microparticles (white bars) for 4 hours before activation with LPS or PAM3CSK4 for 24 hours. Cells were washed and lysed. RNA was isolated and fibronectin levels were measured via qPCR (<b>A</b>), indicating PPARγ MP increased fibronectin expression from activated cells. <b>B</b>, Integrins ITGA5 and ITGAV were also measured by qPCR, showing THP-1 cells treated with PPARγ MP had the highest expression of the fibronectin receptor, ITGA5, but no significant changes were seen with the vitronectin receptor, ITGAV. <b>C–E</b>, THP-1 cells were directly transduced with a control (GFP THP1; grey bars) or a PPARγ-overexpressing lentivirus (PPARγ THP1; black bars) and expanded for one week. Cells were then plated and left unactivated, or stimulated with LPS or PAM3CSK4 for 24 hours at which point suspension cells were removed and the wells were washed three times in phosphate buffered saline. <b>C</b>, Adherent cells were visualized and counted with an inverted microscope, and cells which overexpressed PPARγ demonstrated increased adherence compared to the control groups. Unactivated transduced THP-1 cells were lysed for mRNA (<b>D</b>) and protein (<b>E</b>) analysis. THP-1 cells that overexpress PPARγ had significant upregulation of fibronectin mRNA and protein as well as ITGA5 protein. Mean values with standard errors represent one of at least 3 experiments. Two-way ANOVA with Tukey's multiple comparison post test (<b>A–C</b>), or Student's T Test (<b>D–E</b>) were performed to determine statistical significance. * indicates (p<0.05).</p

Culture-derived microparticles engineered to contain PPARγ are internalized by THP-1 cells.

<p>Microparticles isolated from Meg-01 cultures were characterized for size by dynamic light scattering. <b>A</b>, Percent of the total number of analyzed particles was graphed showing the engineered microparticles have diameters ranging from 0.1–1 µm. <b>B</b>, Control MP (1) or microparticles that contain PPARγ (PPARγ MP; 2) were lysed and analyzed via western blot for PPARγ protein expression (arrow). <b>C</b>, Expression of mRNA transcripts were compared between Control MP or PPARγ MP, where red dots indicate transcripts that are more abundant in PPARγ MP, and green dots indicate transcripts that are more abundant in Control MP. <b>D</b>, Select transcript targets with arrows highlight comparative differences between the microparticle groups. * signifies transcripts were undetectable in Control MP, <b>^†</b> signifies transcripts that were undetectable in PPARγ MP. <b>E</b>, Green fluorescent protein (GFP)-positive microparticles were added to THP-1 culture for six hours before the cells were collected, fixed, cytospun, and visualized by microscopy. Microparticles (green) are associated with the cell surface (arrows) or within the cytoplasm (circle). <b>F</b>, Microparticle internalization by THP-1 cells with sub-saturating numbers of microparticles was measured by flow cytometry and graphed. Basal microparticle internalization rose from approximately 33% of cells (grey horizontal bar) to 60% with increasing doses of PAM3CSK4 (black solid line) or PMA (grey dash line).</p