scholarly journals Expression of Endogenous Putative TSH Binding Protein in Orbit

2021 ◽  
Vol 43 (3) ◽  
pp. 1794-1804
Author(s):  
Mohd Shazli Draman ◽  
Fiona Grennan-Jones ◽  
Peter Taylor ◽  
Ilaria Muller ◽  
Sam Evans ◽  
...  
Keyword(s):  
Cyclic Amp ◽  
Western Blot ◽  
Limit Of Detection ◽  
Culture Media ◽  
Ex Vivo ◽  
Luciferase Reporter ◽  
Positive Signal ◽  
Differentiated Cells ◽  
Differentiation Protocol ◽  
Graves Orbitopathy

Thyroid stimulating antibodies (TSAB) cause Graves’ disease and contribute to Graves’ Orbitopathy (GO) pathogenesis. We hypothesise that the presence of TSH binding proteins (truncated TSHR variants (TSHRv)) and/or nonclassical ligands such as thyrostimulin (α2β5) might provide a mechanism to protect against or exacerbate GO. We analysed primary human orbital preadipocyte-fibroblasts (OF) from GO patients and people free of GO (non-GO). Transcript (QPCR) and protein (western blot) expression levels of TSHRv were measured through an adipogenesis differentiation process. Cyclic-AMP production by TSHR activation was studied using luciferase-reporter and RIA assays. After differentiation, TSHRv levels in OF from GO were significantly higher than non-GO (p = 0.039), and confirmed in ex vivo analysis of orbital adipose samples. TSHRv western blot revealed a positive signal at 46 kDa in cell lysates and culture media (CM) from non-GO and GO-OF. Cyclic-AMP decreased from basal levels when OF were stimulated with TSH or Monoclonal TSAB (M22) before differentiation protocol, but increased in differentiated cells, and was inversely correlated with the TSHRv:TSHR ratio (Spearman correlation: TSH r = −0.55, p = 0.23, M22 r = 0.87, p = 0.03). In the bioassay, TSH/M22 induced luciferase-light was lower in CM from differentiated GO-OF than non-GO, suggesting that secreted TSHRv had neutralised their effects. α2 transcripts were present but reduced during adipogenesis (p < 0.005) with no difference observed between non-GO and GO. β5 transcripts were at the limit of detection. Our work demonstrated that TSHRv transcripts are expressed as protein, are more abundant in GO than non-GO OF and have the capacity to regulate signalling via the TSHR.

The pseudogene PTENP1 regulates smooth muscle cells as a competing endogenous RNA

2019 ◽  
Vol 133 (13) ◽  
pp. 1439-1455 ◽  
Author(s):  
Yanxian Lai ◽  
Jianyong Li ◽  
Lintao Zhong ◽  
Xiang He ◽  
Xiaoyun Si ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Western Blot ◽  
Ex Vivo ◽  
Pten Expression ◽  
Luciferase Reporter ◽  
Pcr Analysis ◽  
Akt Phosphorylation ◽  
Proliferation And Apoptosis ◽  
Induced Apoptosis

AbstractThe long non-coding RNA (lncRNA) PTENP1 is a pseudogene of phosphatase and tensin homologue deleted on chromosome ten (PTEN), has been implicated in smooth muscle cell (SMC) proliferation and apoptosis. PTENP1 is the pseudogene of PTEN. However, it is unclear whether and how PTENP1 functions in the proliferation and apoptosis of human aortic SMCs (HASMCs). Here, we hypothesised that PTENP1 inhibits HASMC proliferation and enhances apoptosis by promoting PTEN expression. PCR analysis and Western blot assays respectively showed that both PTENP1 and PTEN were up-regulated in human aortic dissection (AD) samples. PTENP1 overexpression significantly increased the protein expression of PTEN, promoted apoptosis and inhibited the proliferation of HASMCs. PTENP1 silencing exhibited the opposite effects and mitigated H2O2-induced apoptosis of HASMCs. In an angiotensin II (Ang II)-induced mouse aortic aneurysm (AA) model, PTENP1 overexpression potentiated aortic SMC apoptosis, exacerbated aneurysm formation. Mechanistically, RNA pull-down assay and a series of luciferase reporter assays using miR-21 mimics or inhibitors identified PTENP1 as a molecular sponge for miR-21 to endogenously compete for the binding between miR-21 and the PTEN transcript, releasing PTEN expression. This finding was further supported by in vitro immunofluorescent evidence showing decreased cell apoptosis upon miR-21 mimic administration under baseline PTENP1 overexpression. Ex vivo rescue of PTEN significantly mitigated the SMC apoptosis induced by PTENP1 overexpression. Finally, Western blot assays showed substantially reduced Akt phosphorylation and cyclin D1 and cyclin E levels with up-regulated PTENP1 in HASMCs. Our study identified PTENP1 as a mediator of HASMC homeostasis and suggests that PTENP1 is a potential target in AD or AA intervention.

scholarly journals Knockdown of long noncoding RNA HOTAIR inhibits osteoarthritis chondrocyte injury by miR-107/CXCL12 axis

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Jipeng Lu ◽  
Zhongxiong Wu ◽  
Ying Xiong
Keyword(s):  
Cell Proliferation ◽  
Western Blot ◽  
Cell Apoptosis ◽  
Noncoding Rna ◽  
Underlying Mechanism ◽  
Joint Disease ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Cxcl12 Expression ◽  
Ecm Degradation

Abstract Background Osteoarthritis (OA) is a joint disease characterized via destruction of cartilage. Chondrocyte damage is associated with cartilage destruction during OA. Long noncoding RNAs (lncRNAs) are implicated in the regulation of chondrocyte damage in OA progression. This study aims to investigate the role and underlying mechanism of lncRNA homeobox antisense intergenic RNA (HOTAIR) in OA chondrocyte injury. Methods Twenty-three OA patients and healthy controls without OA were recruited. Chondrocytes were isolated from OA cartilage tissues. HOTAIR, microRNA-107 (miR-107) and C-X-C motif chemokine ligand 12 (CXCL12) levels were measured by quantitative real-time polymerase chain reaction and western blot. Cell proliferation, apoptosis and extracellular matrix (ECM) degradation were measured using cell counting kit-8, flow cytometry and western blot. The target interaction was explored by bioinformatics, luciferase reporter and RNA immunoprecipitation assays. Results HOTAIR expression was enhanced, and miR-107 level was reduced in OA cartilage samples. HOTAIR overexpression inhibited cell proliferation, but induced cell apoptosis and ECM degradation in chondrocytes. HOTAIR knockdown caused an opposite effect. MiR-107 was sponged and inhibited via HOTAIR, and knockdown of miR-107 mitigated the effect of HOTAIR silence on chondrocyte injury. CXCL12 was targeted by miR-107. CXCL12 overexpression attenuated the roles of miR-107 overexpression or HOTAIR knockdown in the proliferation, apoptosis and ECM degradation. CXCL12 expression was decreased by HOTAIR silence, and restored by knockdown of miR-107. Conclusion HOTAIR knockdown promoted chondrocyte proliferation, but inhibited cell apoptosis and ECM degradation in OA chondrocytes by regulating the miR-107/CXCL12 axis.

scholarly journals Mesenchymal stem cell-originated exosomal lncRNA HAND2-AS1 impairs rheumatoid arthritis fibroblast-like synoviocyte activation through miR-143-3p/TNFAIP3/NF-κB pathway

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Yuhua Su ◽  
Yajing Liu ◽  
Chao Ma ◽  
Chunxiao Guan ◽  
Xiufen Ma ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Tumor Necrosis Factor ◽  
Western Blot ◽  
Tumor Necrosis ◽  
Cell Counting ◽  
Biological Behavior ◽  
Luciferase Reporter ◽  
Enzyme Linked Immunosorbent Assay ◽  
Binding Interaction ◽  
Necrosis Factor

Abstract Background Long non-coding RNA heart and neural crest derivatives expressed 2-antisense RNA 1 (HAND2-AS1) was found to be elevated in rheumatoid arthritis (RA) fibroblast-like synoviocytes (RA-FLSs). However, whether HAND2-AS1 functions as an exosomal lncRNA related to mesenchymal stem cells (MSCs) in RA progression is unknown. Methods The expression of HAND2-AS1, microRNA (miR)-143-3p, and tumor necrosis factor alpha-inducible protein 3 (TNFAIP3) was detected using quantitative real-time polymerase chain reaction and Western blot. Cell proliferation, apoptosis, migration, and invasion were detected using cell counting kit-8, flow cytometry, and wound healing and transwell assays. The levels of tumor necrosis factor-α (TNF-α) and interleukins (IL)-6 were analyzed using enzyme-linked immunosorbent assay. The level of phosphorylated-p65 was examined by Western blot. The binding interaction between miR-143-3p and HAND2-AS1 or TNFAIP3 was confirmed by the dual-luciferase reporter and RIP assays. Exosomes were isolated by ultracentrifugation and qualified by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blot. Results HAND2-AS1 was lowly expressed in RA synovial tissues, and HAND2-AS1 re-expression suppressed the proliferation, motility, and inflammation and triggered the apoptosis in RA-FLSs via the inactivation of NF-κB pathway. Mechanistically, HAND2-AS1 directly sponged miR-143-3p and positively regulated TNFAIP3 expression, the target of miR-143-3p. Moreover, the effects of HAND2-AS1 on RA-FLSs were partially attenuated by miR-143-3p upregulation or TNFAIP3 knockdown. HAND2-AS1 could be packaged into hMSC-derived exosomes and absorbed by RA-FLSs, and human MSC-derived exosomal HAND2-AS1 also repressed above malignant biological behavior of RA-FLSs. Conclusion MSC-derived exosomes participated in the intercellular transfer of HAND2-AS1 and suppressed the activation of RA-FLSs via miR-143-3p/TNFAIP3/NF-κB pathway, which provided a novel insight into the pathogenesis and treatment of RA.

Effect of dexamethasone on the expression of MMPs, adenosine A1 receptors and NFKB by human trabecular meshwork cells

2020 ◽  
Vol 0 (0) ◽  
Author(s):  
Normie Aida Mohd Nasir ◽  
Renu Agarwal ◽  
Anna Krasilnikova ◽  
Siti Hamimah Sheikh Abdul Kadir ◽  
Igor Iezhitsa
Keyword(s):  
Extracellular Matrix ◽  
Western Blot ◽  
Trabecular Meshwork ◽  
Culture Media ◽  
Smooth Muscle Actin ◽  
Adenosine A1 Receptors ◽  
Trabecular Meshwork Cells ◽  
Aqueous Humor Dynamics ◽  
Nfkb Activation ◽  
Adenosine A1

AbstractObjectivesSteroid-induced ocular hypertension and glaucoma are associated with extracellular matrix remodeling at the trabecular meshwork (TM) of the eye due to reduced secretion of matrix metalloproteinases (MMPs), a family of enzymes regulating extracellular matrix proteolysis. Several biological functions of steroids are known to involve regulation of adenosine A1 receptors (A1AR) and nuclear factor kappa B (NFKB). Since MMPs expression in TM has been shown to be regulated by A1AR as well as transcription factors, it is likely that dexamethasone-induced changes in aqueous humor dynamics involve reduced MMP and A1AR expression and reduced NFKB activation. Hence, the current study investigated the association of dexamethasone-induced reduction in MMP secretion with reduced NFKB activation and A1AR expression.MethodsHuman trabecular meshwork cells (HTMCs) were characterized by estimating myocilin and alpha smooth muscle actin expression and then were treated with dexamethasone 100 nM for 2, 5 and 7 days. The MMP secretion was estimated in culture media using Western blot. Immunocytochemistry (ICC) and ELISA were done to investigate the effect of dexamethasone on NFKB phosphorylation. A1AR expression in HTMCs was determined using Western blot and ELISA.ResultsDexamethasone caused a significant reduction in both MMP-2 and -9 expression compared to untreated group after five and seven days but not after two days of culture. Significantly reduced phosphorylated NFKB and A1AR protein levels were detected in dexamethasone treated compared to vehicle treated HTMCs after five days of culture.ConclusionsDexamethasone reduces MMP-2 and -9 secretion by HTMCs and this effect of dexamethasone is associated with reduced NFKB phosphorylation and A1AR expression.

scholarly journals Pharmacological ablation of astrocytes reduces Aβ degradation and synaptic connectivity in an ex vivo model of Alzheimer’s disease

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Nicola Davis ◽  
Bibiana C. Mota ◽  
Larissa Stead ◽  
Emily O. C. Palmer ◽  
Laura Lombardero ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Culture Media ◽  
Ex Vivo ◽  
Wild Type ◽  
Insulin Degrading Enzyme ◽  
Ex Vivo Model ◽  
Model Of Alzheimer’S Disease ◽  
Aβ Clearance ◽  
5Xfad Mice

Abstract Background Astrocytes provide a vital support to neurons in normal and pathological conditions. In Alzheimer’s disease (AD) brains, reactive astrocytes have been found surrounding amyloid plaques, forming an astrocytic scar. However, their role and potential mechanisms whereby they affect neuroinflammation, amyloid pathology, and synaptic density in AD remain unclear. Methods To explore the role of astrocytes on Aβ pathology and neuroinflammatory markers, we pharmacologically ablated them in organotypic brain culture slices (OBCSs) from 5XFAD mouse model of AD and wild-type (WT) littermates with the selective astrocytic toxin L-alpha-aminoadipate (L-AAA). To examine the effects on synaptic circuitry, we measured dendritic spine number and size in OBCSs from Thy-1-GFP transgenic mice incubated with synthetic Aβ42 or double transgenics Thy-1-GFP/5XFAD mice treated with LAAA or vehicle for 24 h. Results Treatment of OBCSs with L-AAA resulted in an increased expression of pro-inflammatory cytokine IL-6 in conditioned media of WTs and 5XFAD slices, associated with changes in microglia morphology but not in density. The profile of inflammatory markers following astrocytic loss was different in WT and transgenic cultures, showing reductions in inflammatory mediators produced in astrocytes only in WT sections. In addition, pharmacological ablation of astrocytes led to an increase in Aβ levels in homogenates of OBCS from 5XFAD mice compared with vehicle controls, with reduced enzymatic degradation of Aβ due to lower neprilysin and insulin-degrading enzyme (IDE) expression. Furthermore, OBSCs from wild-type mice treated with L-AAA and synthetic amyloid presented 56% higher levels of Aβ in culture media compared to sections treated with Aβ alone, concomitant with reduced expression of IDE in culture medium, suggesting that astrocytes contribute to Aβ clearance and degradation. Quantification of hippocampal dendritic spines revealed a reduction in their density following L-AAA treatment in all groups analyzed. In addition, pharmacological ablation of astrocytes resulted in a decrease in spine size in 5XFAD OBCSs but not in OBCSs from WT treated with synthetic Aβ compared to vehicle control. Conclusions Astrocytes play a protective role in AD by aiding Aβ clearance and supporting synaptic plasticity.

scholarly journals NEAT1 aggravates sepsis-induced acute kidney injury by sponging miR-22-3p

Open Medicine ◽  
2020 ◽  
Vol 15 (1) ◽  
pp. 333-342
Author(s):  
Yawei Feng ◽  
Jun Liu ◽  
Ranliang Wu ◽  
Peng Yang ◽  
Zhiqiang Ye ◽  
...  
Keyword(s):  
Acute Kidney Injury ◽  
Western Blot ◽  
Cell Apoptosis ◽  
Noncoding Rna ◽  
Cell Injury ◽  
Kidney Injury ◽  
Inflammatory Factors ◽  
Luciferase Reporter ◽  
Enzyme Linked Immunosorbent Assay ◽  
Western Blot Assay

AbstractBackground and aimAcute kidney injury (AKI) is a common complication of sepsis. Long noncoding RNA nuclear-enriched abundant transcript 1 (NEAT1) plays a vital role in various diseases, including AKI. This study aimed to investigate the function and mechanism of NEAT1 in sepsis-induced AKI.Materials and methodsA septic AKI model was established by treating HK-2 cells with lipopolysaccharide (LPS). The levels of NEAT1 and miR-22-3p were measured by quantitative real-time PCR. Cell apoptosis was assessed by flow cytometry. The levels of apoptosis-related protein and autophagy-related factors were examined by the western blot assay. An enzyme-linked immunosorbent assay was used to calculate the contents of inflammatory factors. The interaction between NEAT1 and miR-22-3p was validated by dual-luciferase reporter assay, RNA immunoprecipitation assay, and RNA pull-down assay. The levels of nuclear factor (NF)-κB pathway-related proteins were evaluated by the western blot assay.ResultsNEAT1 was upregulated, while miR-22-3p was downregulated in patients with sepsis and in LPS-stimulated HK-2 cells. LPS treatment triggered cell apoptosis, autophagy, and inflammatory response in HK-2 cells. NEAT1 knockdown attenuated LPS-induced cell injury. NEAT1 modulated LPS-triggered cell injury by targeting miR-22-3p. Furthermore, NEAT1 regulated the NF-κB pathway by modulating miR-22-3p.ConclusionDepletion of NEAT1 alleviated sepsis-induced AKI via regulating the miR-22-3p/NF-κB pathway.

scholarly journals Knockdown of lncRNA X inactive specific transcript (XIST) radiosensitizes non-small cell lung cancer (NSCLC) cells through regulation of miR-16-5p/WEE1 G2 checkpoint kinase (WEE1) axis

2021 ◽  
Vol 35 ◽  
pp. 205873842096608
Author(s):  
Ran Du ◽  
Feng Jiang ◽  
Yanhua Yin ◽  
Jinfen Xu ◽  
Xia Li ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Western Blot ◽  
Small Cell ◽  
Luciferase Reporter ◽  
Small Cell Lung ◽  
Luciferase Reporter Gene ◽  
G2 Checkpoint ◽  
Qrt Pcr ◽  
Specific Transcript

Long non-coding RNA (lncRNA) X inactive specific transcript (XIST) is reported to play an oncogenic role in non-small cell lung cancer (NSCLC). However, the role of XIST in regulating the radiosensitivity of NSCLC cells remains unclear. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expressions of XIST and miR-16-5p in NSCLC in tissues and cells, and Western blot was used to assess the expression of WEE1 G2 checkpoint kinase (WEE1). Cell counting kit-8 (CCK-8), colony formation and flow cytometry assays were used to determine cell viability and apoptosis after NSCLC cells were exposed to different doses of X-rays. The interaction between XIST and miR-16-5p was confirmed by StarBase database, qRT-PCR and dual-luciferase reporter gene assays. TargetScan database was used to predict WEE1 as a target of miR-16-5p, and their targeting relationship was further validated by Western blot, qRT-PCR and dual-luciferase reporter gene assays. XIST was highly expressed in both NSCLC tissue and cell lines, and knockdown of XIST repressed NSCLC cell viability and cell survival, and facilitated apoptosis under the irradiation. MiR-16-5p was a target of XIST, and rescue experiments demonstrated that miR-16-5p inhibitors could reverse the role of XIST knockdown on radiosensitivity in NSCLC cells. WEE1 was validated as a target gene of miR-16-5p, and WEE1 could be negatively regulated by XIST. XIST promotes the radioresistance of NSCLC cells by regulating the expressions of miR-16-5p and WEE1, which can be a novel target for NSCLC therapy.

scholarly journals miR-299-3p suppresses cell progression and induces apoptosis by downregulating PAX3 in gastric cancer

2021 ◽  
Vol 16 (1) ◽  
pp. 266-276
Author(s):  
Zhenfen Wang ◽  
Qing Liu ◽  
Ping Huang ◽  
Guohao Cai
Keyword(s):  
Gastric Cancer ◽  
Cell Proliferation ◽  
Western Blot ◽  
Luciferase Reporter ◽  
Reporter System ◽  
Western Blot Assay ◽  
Normal Tissues ◽  
Cell Progression ◽  
Luciferase Reporter System ◽  
Proliferation And Invasion

Abstract Gastric cancer (GC) is ranked the fourth leading cause of cancer-related death, with an over 75% mortality rate worldwide. In recent years, miR-299-3p has been identified as a biomarker in multiple cancers, such as acute promyelocytic leukemia, thyroid cancer, and lung cancer. However, the regulatory mechanism of miR-299-3p in GC cell progression is still largely unclear. Cell viability and apoptosis tests were performed by CCK8 and flow cytometry assay, respectively. Transwell assay was recruited to examine cell invasion ability. The interaction between miR-299-3p and PAX3 was determined by the luciferase reporter system. PAX3 protein level was evaluated by western blot assay. The expression of miR-299-3p was downregulated in GC tissues and cell lines (MKN-45, AGS, and MGC-803) compared with the normal tissues and cells. Besides, overexpression of miR-299-3p significantly suppressed proliferation and invasion and promoted apoptosis in GC. Next, we clarified that PAX3 expression was regulated by miR-299-3p using a luciferase reporter system, qRT-PCR, and western blot assay. Additionally, downregulation of PAX3 repressed GC cell progression. The rescue experiments indicated that restoration of PAX3 inversed miR-299-3p-mediated inhibition on cell proliferation and invasion. miR-299-3p suppresses cell proliferation and invasion as well as induces apoptosis by regulating PAX3 expression in GC, representing desirable biomarkers for GC diagnosis and therapy.

scholarly journals Intestinal crypt derived enteroid coculture in presence of peristaltic longitudinal muscle myenteric plexus

2020 ◽  
Author(s):  
Daniel E Levin ◽  
Arabinda Mandal ◽  
Mark A Fleming ◽  
Katherine H Bae ◽  
Brielle Gerry ◽  
...  
Keyword(s):  
Myenteric Plexus ◽  
Longitudinal Muscle ◽  
Culture Media ◽  
Ex Vivo ◽  
Enteric Neurons ◽  
Intestinal Cell ◽  
Cell Populations ◽  
Complex Interactions ◽  
Proliferation And Differentiation ◽  
Intestinal Peristalsis

Abstract The role of enteric neurons in driving intestinal peristalsis has been known for over a century. However, in recent decades, scientists have begun to unravel additional complex interactions between this nerve plexus and other cell populations in the intestine. Investigations into these potential interactions is complicated by a paucity of tractable models of these cellular relationships. Here, we describe a novel technique for ex vivo coculture of enteroids, so called “mini-guts,” in juxtaposition to the longitudinal muscle myenteric plexus (LMMP). Key to this system, we developed a LMMP culture media that: 1) allows the LMMP to maintain ex vivo peristalsis for 2 weeks along with proliferation of neurons, glia, smooth muscle and fibroblast cells, and 2) supports the proliferation and differentiation of the intestinal stem cells into enteroids complete with epithelial enterocytes, Paneth cells, goblet cells and enteroendocrine cells. Importantly, this technique identifies a culture condition that supports both the metabolic needs of intestinal epithelium as well as neuronal elements, demonstrating the feasibility of maintaining these two populations in a single culture system. This sets the stage for experiments to better define the regulatory interactions of these two important intestinal cell populations.

scholarly journals PPARγ inhibits airway epithelial cell inflammatory response through a MUC1-dependent mechanism

2012 ◽  
Vol 302 (7) ◽  
pp. L679-L687 ◽  
Author(s):  
Yong Sung Park ◽  
Erik P. Lillehoj ◽  
Kosuke Kato ◽  
Choon Sik Park ◽  
Kwang Chul Kim
Keyword(s):  
Epithelial Cells ◽  
Culture Media ◽  
A549 Cells ◽  
Luciferase Reporter ◽  
Muc1 Expression ◽  
Mrna Levels ◽  
Airway Epithelial ◽  
Pparγ Agonist ◽  
Tnf Α ◽  
Primary Mouse

This study was conducted to examine the relationship between the peroxisome proliferator-associated receptor-γ (PPARγ) and MUC1 mucin, two anti-inflammatory molecules expressed in the airways. Treatment of A549 lung epithelial cells or primary mouse tracheal surface epithelial (MTSE) cells with phorbol 12-myristate 13-acetate (PMA) increased the levels of tumor necrosis factor (TNF)-α in cell culture media compared with cells treated with vehicle alone. Overexpression of MUC1 in A549 cells decreased PMA-stimulated TNF-α levels, whereas deficiency of Muc1 expression in MTSE cells from Muc1 null mice increased PMA-induced TNF-α levels. Treatment of A549 or MTSE cells with the PPARγ agonist troglitazone (TGN) blocked the ability of PMA to stimulate TNF-α levels. However, the effect of TGN required the presence of MUC1/Muc1, since no differences in TNF-α levels were seen between PMA and PMA plus TGN in MUC1/Muc1-deficient cells. Similarly, whereas TGN decreased interleukin-8 (IL-8) levels in culture media of MUC1-expressing A549 cells treated with Pseudomonas aeruginosa strain K (PAK), no differences in IL-8 levels were seen between PAK and PAK plus TGN in MUC1-nonexpressing cells. EMSA confirmed the presence of a PPARγ-binding element in the MUC1 gene promoter. Finally, TGN treatment of A549 cells increased MUC1 promoter activity measured using a MUC1-luciferase reporter gene, augmented MUC1 mRNA levels by quantitative RT-PCR, and enhanced MUC1 protein expression by Western blot analysis. These combined data are consistent with the hypothesis that PPARγ stimulates MUC1/Muc1 expression, thereby blocking PMA/PAK-induced TNF-α/IL-8 production by airway epithelial cells.

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