Acetyl-CoA carboxylase inhibition regulates microtubule dynamics and intracellular transport in cystic fibrosis epithelial cells

The use of high-dose ibuprofen as an anti-inflammatory therapy in cystic fibrosis (CF) has been shown to be an effective intervention although use is limited due to potential adverse events. Identifying the mechanism of ibuprofen efficacy would aid in the development of new therapies that avoid these adverse events. Previous findings demonstrated that ibuprofen treatment restores the regulation of microtubule dynamics in CF epithelial cells through a 5′-adenosine monophosphate-activated protein kinase (AMPK)-dependent mechanism. The goal of this study is to define the AMPK pathway that leads to microtubule regulation. Here, it is identified that inhibition of acetyl-CoA carboxylase (ACC) is the key step in mediating the AMPK effect. ACC inhibition with 5-(tetradecyloxy)-2-furoic acid (TOFA) increases microtubule reformation rates in cultured and primary CF epithelial cells to wild-type (WT) rates. TOFA treatment also restores microtubule-dependent distribution of cholesterol and Rab7-positive organelles, as well as reduces expression of the proinflammatory signaling molecule RhoA to WT levels. ACC activation with citrate replicates these CF phenotypes in WT cells further supporting the role of AMPK signaling through ACC as a key mediator in CF cell signaling. It is concluded that ACC inhibition is the key step in the efficacy of AMPK activation at the cellular level and could represent a novel site of therapeutic intervention to address inflammation in CF.

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Ibuprofen regulation of microtubule dynamics in cystic fibrosis epithelial cells

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00126.2016 ◽

2016 ◽

Vol 311 (2) ◽

pp. L317-L327 ◽

Cited By ~ 12

Author(s):

Sharon M. Rymut ◽

Claire M. Kampman ◽

Deborah A. Corey ◽

Tori Endres ◽

Calvin U. Cotton ◽

...

Keyword(s):

Cystic Fibrosis ◽

Epithelial Cells ◽

Intracellular Transport ◽

Pediatric Population ◽

Microtubule Dynamics ◽

Cyclooxygenase Inhibitors ◽

High Dose ◽

Cell Periphery ◽

Wild Type ◽

Compound C

High-dose ibuprofen, an effective anti-inflammatory therapy for the treatment of cystic fibrosis (CF), has been shown to preserve lung function in a pediatric population. Despite its efficacy, few patients receive ibuprofen treatment due to potential renal and gastrointestinal toxicity. The mechanism of ibuprofen efficacy is also unclear. We have previously demonstrated that CF microtubules are slower to reform after depolymerization compared with respective wild-type controls. Slower microtubule dynamics in CF cells are responsible for impaired intracellular transport and are related to inflammatory signaling. Here, it is identified that high-dose ibuprofen treatment in both CF cell models and primary CF nasal epithelial cells restores microtubule reformation rates to wild-type levels, as well as induce extension of microtubules to the cell periphery. Ibuprofen treatment also restores microtubule-dependent intracellular transport monitored by measuring intracellular cholesterol transport. These effects are specific to ibuprofen as other cyclooxygenase inhibitors have no effect on these measures. Effects of ibuprofen are mimicked by stimulation of AMPK and blocked by the AMPK inhibitor compound C. We conclude that high-dose ibuprofen treatment enhances microtubule formation in CF cells likely through an AMPK-related pathway. These findings define a potential mechanism to explain the efficacy of ibuprofen therapy in CF.

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3,5-Dicaffeoyl-Epi-Quinic Acid Isolated from Edible Halophyte Atriplex gmelinii Inhibits Adipogenesis via AMPK/MAPK Pathway in 3T3-L1 Adipocytes

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2018/8572571 ◽

2018 ◽

Vol 2018 ◽

pp. 1-8 ◽

Cited By ~ 6

Author(s):

Jung Hwan Oh ◽

Jung Im Lee ◽

Fatih Karadeniz ◽

Youngwan Seo ◽

Chang-Suk Kong

Keyword(s):

Protein Kinase ◽

Molecular Mechanisms ◽

Mapk Pathway ◽

Health Benefits ◽

Quinic Acid ◽

Adenosine Monophosphate ◽

Signal Pathways ◽

Acetyl Coa Carboxylase ◽

Acetyl Coa ◽

Related Factors

Atriplex gmelinii is an edible halophyte that has been suggested to possess various health benefits. In the present study, 3,5-dicaffeoyl-epi-quinic acid (DEQA) isolated from A. gmelinii was tested for its ability to prevent adipogenesis in 3T3-L1 cells. Also, the molecular mechanisms by which DEQA affects differentiation of 3T3-L1 cells were investigated. The introduction of DEQA to differentiating 3T3-L1 preadipocytes resulted in suppressed adipogenesis and lowered expression of adipogenesis-related factors, PPARγ, C/EBPα, and SREBP-1c. Treatment of 3T3-L1 adipocytes with DEQA notably decreased the levels of phosphorylated p38, ERK, and JNK. In addition, presence of DEQA upregulated the levels of both inactive and phosphorylated adenosine monophosphate-activated protein kinase (AMPK) and its substrate, acetyl-CoA carboxylase (ACC). Taken together, current results indicated that DEQA exhibited a significant antiadipogenesis activity by activation of AMPK and downregulation of MAPK signal pathways in 3T3-L1 preadipocytes.

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Phosphorylation of Acetyl-CoA Carboxylase by AMPK Reduces Renal Fibrosis and Is Essential for the Anti-Fibrotic Effect of Metformin

Journal of the American Society of Nephrology ◽

10.1681/asn.2018010050 ◽

2018 ◽

Vol 29 (9) ◽

pp. 2326-2336 ◽

Cited By ~ 27

Author(s):

Mardiana Lee ◽

Marina Katerelos ◽

Kurt Gleich ◽

Sandra Galic ◽

Bruce E. Kemp ◽

...

Keyword(s):

Fatty Acid ◽

Epithelial Cells ◽

Lipid Accumulation ◽

Acid Oxidation ◽

Acetyl Coa Carboxylase ◽

Tubular Epithelial Cells ◽

Acetyl Coa ◽

Expression Of Genes ◽

Energy Sensing ◽

Unilateral Ureteric Obstruction

BackgroundExpression of genes regulating fatty acid metabolism is reduced in tubular epithelial cells from kidneys with tubulointerstitial fibrosis (TIF), thus decreasing the energy produced by fatty acid oxidation (FAO). Acetyl-CoA carboxylase (ACC), a target for the energy-sensing AMP-activating protein kinase (AMPK), is the major controller of the rate of FAO within cells. Metformin has a well described antifibrotic effect, and increases phosphorylation of ACC by AMPK, thereby increasing FAO.MethodsWe evaluated phosphorylation of ACC in cell and mouse nephropathy models, as well as the effects of metformin administration in mice with and without mutations that reduce ACC phosphorylation.ResultsReduced phosphorylation of ACC on the AMPK site Ser79 occurred in both tubular epithelial cells treated with folate to mimic cellular injury and in wild-type (WT) mice after induction of the folic acid nephropathy model. When this effect was exaggerated in mice with knock-in (KI) Ser to Ala mutations of the phosphorylation sites in ACC, lipid accumulation and fibrosis increased significantly compared with WT. The effect of ACC phosphorylation on fibrosis was confirmed in the unilateral ureteric obstruction model, which showed significantly increased lipid accumulation and fibrosis in the KI mice. Metformin use was associated with significantly reduced fibrosis and lipid accumulation in WT mice. In contrast, in the KI mice, the drug was associated with worsened fibrosis.ConclusionsThese data indicate that reduced phosphorylation of ACC after renal injury contributes to the development of TIF, and that phosphorylation of ACC is required for metformin’s antifibrotic action in the kidney.

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DIBI, a novel polymeric iron chelator modulates IL-6 and IL-8 secretion from Cystic Fibrosis airway epithelial cells in response to endotoxin induction

Journal of Cellular Biotechnology ◽

10.3233/jcb-209011 ◽

2020 ◽

pp. 1-10

Author(s):

Maral Aali ◽

Alexa Caldwell ◽

Audrey Li ◽

Bruce Holbein ◽

Valerie Chappe ◽

...

Keyword(s):

Cystic Fibrosis ◽

Epithelial Cells ◽

Chronic Inflammation ◽

Therapeutic Potential ◽

Iron Chelation ◽

Iron Chelator ◽

Airway Epithelial Cells ◽

High Dose ◽

Dependent Manner ◽

Lps Challenge

Iron chelators have been utilized clinically to treat patients with iron overload conditions. There is a growing body of evidence linking iron dysregulation and reactive oxygen species (ROS) overproduction as underlying factors in Cystic Fibrosis (CF) disease. The chronic inflammation can lead to progressive airway destruction. Alleviation of this chronic inflammation is a potential target for CF treatment and thus, this research investigated the dose-response effects of DIBI, a novel iron chelator, on inflammation in CF nasal epithelial cells. Polarized CF cells were stimulated with, lipopolysaccharide (LPS), co-treated with DIBI (LPS+DIBI), or DIBI alone (DIBI). We demonstrated that DIBI modulated the release of IL-6 and IL-8 in CF cells in a dose-dependent manner. Reduction of extracellular iron with the lower doses of DIBI (25 and 50μM), increased IL-6 secretion in non-induced cells. LPS challenge increased IL-6 and IL-8 secretion which was suppressed by high dose (200μM) DIBI administration. This study demonstrates the therapeutic potential of iron chelation therapy to treat the dysregulation of the immune response in CF patients.

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High dose everolimus promotes a pro-inflammatory phenotype and facilitates the epithelial to mesenchymal transition of primary bronchial epithelial cells isolated from cystic fibrosis patients.

10.21203/rs.3.rs-46751/v1 ◽

2020 ◽

Author(s):

Simona Granata ◽

Gloria Santoro ◽

Valentina Masola ◽

Paola Pontrelli ◽

Fabio Sallustio ◽

...

Keyword(s):

Cystic Fibrosis ◽

Epithelial Cells ◽

Pathway Analysis ◽

Epithelial To Mesenchymal Transition ◽

Bronchial Epithelial Cells ◽

High Dose ◽

Bronchial Epithelial ◽

Mesenchymal Transition ◽

Primary Bronchial Epithelial Cells ◽

Epithelial Resistance

Abstract Background: Lung transplantation is still the best therapeutic option for cystic fibrosis (CF) patients, but, unfortunately, immunosuppressive therapies, often employed at high dosages to avoid acute rejection, may induce severe complications. In particular, patients treated with high dose of mammalian target of rapamycin inhibitors (mTOR-Is) may experience lung fibrosis (including bronchiolitis obliterans-organizing pneumonia). Although epithelial to mesenchymal transition (EMT) of airway cells has a central role in this process, the complete biological machinery is not completely clarified.Methods: In order to improve our knowledge on this process, primary bronchial epithelial cells carrying F508del mutation were treated with 5 and 100 nM everolimus (EVE) for 24 hours. Subsequently, RNA was hybridized to the Human HT-12 v3 Expression BeadChip (Illumina). Real-Time PCR was, then, used to validate microarray results and to measure major EMT biomarkers. Trans-epithelial resistance was measured by Millicell-ERS ohmmeter.Results: High dosage EVE induced a significant up-regulation of 42 genes and a down-regulation of 12 genes. After pathway analysis by Gene Set Enrichment Analysis and Ingenuity Pathway Analysis, we found that most of them were implicated in the pro-inflammatory pathway. Real-Time PCR validated these results and revealed that, in addition to pro-inflammatory genes (IL-1α, IL-8, Pim-1 Oncogene), EVE at high dosage was able to up-regulate major EMT biomarkers (such as: alpha-smooth muscle actin, connective tissue growth factor and metalloproteinase 12). In lung, EMT is the convergence point between inflammation and the progression of fibrotic damage. Additionally, EVE at this dosage reduced the trans-epithelial resistance (altering tight junction strength). In contrast, lower EVE did not trigger similar effects.Conclusions: We demonstrated that high dose EVE may trigger a pro-inflammatory/fibrotic biological machinery in bronchial epithelial cells from CF patients. Our results, although obtained in vitro, suggest that solid organ transplant recipients affected by CF should be treated with the lowest possible dose of mTOR inhibitors to minimize/avoid the onset and development of lung complications. In vivo studies might be useful to confirm our hypothesis.

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Resveratrol restores intracellular transport in cystic fibrosis epithelial cells

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00006.2020 ◽

2020 ◽

Vol 318 (6) ◽

pp. L1145-L1157 ◽

Cited By ~ 1

Author(s):

Binyu Lu ◽

Deborah A. Corey ◽

Thomas J. Kelley

Keyword(s):

Cystic Fibrosis ◽

Animal Models ◽

Epithelial Cells ◽

Intracellular Transport ◽

Peroxisome Proliferator ◽

Cell Models ◽

Peroxisome Proliferator Activated Receptor ◽

Ability To Act ◽

Microtubule Formation ◽

Inflammatory Regulation

We have demonstrated previously that intracellular transport is impaired in cystic fibrosis (CF) epithelial cells. This impairment is related to both growth and inflammatory regulation in CF cell and animal models. Understanding how transport in CF cells is regulated and identifying means to manipulate that regulation are key to identifying new therapies that can address key CF phenotypes. It was hypothesized that resveratrol could replicate these benefits since it interfaces with multiple pathways identified to affect microtubule regulation in CF. It was found that resveratrol treatment significantly restored intracellular transport as determined by monitoring both cholesterol distribution and the distribution of rab7-positive organelles in CF cells. This restoration of intracellular transport is due to correction of both microtubule formation rates and microtubule acetylation in cultured CF cell models and primary nasal epithelial cells. Mechanistically, the effect of resveratrol on microtubule regulation and intracellular transport was dependent on peroxisome proliferator-activated receptor-γ signaling and its ability to act as a pan-histone deacetylase (HDAC) inhibitor. Resveratrol represents a candidate compound with known anti-inflammatory properties that can restore both microtubule formation and acetylation in CF epithelial cells.

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EG-VEGF, BV8, and their receptor expression in human bronchi and their modification in cystic fibrosis: Impact of CFTR mutation (delF508)

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00382.2014 ◽

2015 ◽

Vol 309 (3) ◽

pp. L314-L322 ◽

Cited By ~ 3

Author(s):

Sylvain Chauvet ◽

Wael Traboulsi ◽

Laura Thevenon ◽

Amal Kouadri ◽

Jean-Jacques Feige ◽

...

Keyword(s):

Cystic Fibrosis ◽

Epithelial Cells ◽

Chloride Channel ◽

Mrna Level ◽

Cellular Level ◽

Receptor Expression ◽

Endocrine Gland ◽

Channel Activity ◽

Endothelial Growth Factor

Enhanced lung angiogenesis has been reported in cystic fibrosis (CF). Recently, two highly homologous ligands, endocrine gland vascular endothelial growth factor (EG-VEGF) and mammalian Bv8, have been described as new angiogenic factors. Both ligands bind and activate two closely related G protein-coupled receptors, the prokineticin receptor (PROKR) 1 and 2. Yet, the expression, regulation, and potential role of EG-VEGF, BV8, and their receptors in normal and CF lung are still unknown. The expression of the receptors and their ligands was examined using molecular, biochemical, and immunocytochemistry analyses in lungs obtained from CF patients vs. control and in normal and CF bronchial epithelial cells. Cystic fibrosis transmembrane conductance regulator (CFTR) activity was evaluated in relation to both ligands, and concentrations of EG-VEGF were measured by ELISA. At the mRNA level, EG-VEGF, BV8, and PROKR2 gene expression was, respectively, approximately five, four, and two times higher in CF lungs compared with the controls. At the cellular level, both the ligands and their receptors showed elevated expressions in the CF condition. Similar results were observed at the protein level. The EG-VEGF secretion was apical and was approximately two times higher in CF compared with the normal epithelial cells. This secretion was increased following the inhibition of CFTR chloride channel activity. More importantly, EG-VEGF and BV8 increased the intracellular concentration of Ca2+ and cAMP and stimulated CFTR-chloride channel activity. Altogether, these data suggest local roles for epithelial BV8 and EG-VEGF in the CF airway peribronchial vascular remodeling and highlighted the role of CFTR activity in both ligand biosynthesis and secretion.

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Reciprocal Regulation of AMPK/SNF1 and Protein Acetylation

International Journal of Molecular Sciences ◽

10.3390/ijms19113314 ◽

2018 ◽

Vol 19 (11) ◽

pp. 3314 ◽

Cited By ~ 12

Author(s):

Ales Vancura ◽

Shreya Nagar ◽

Pritpal Kaur ◽

Pengli Bu ◽

Madhura Bhagwat ◽

...

Keyword(s):

Histone Deacetylases ◽

Mammalian Cells ◽

Adenosine Monophosphate ◽

Cellular Level ◽

Yeast Cells ◽

Protein Acetylation ◽

Acetyl Coa ◽

Reciprocal Regulation ◽

Atp Production ◽

Upstream Kinase

Adenosine monophosphate (AMP)-activated protein kinase (AMPK) serves as an energy sensor and master regulator of metabolism. In general, AMPK inhibits anabolism to minimize energy consumption and activates catabolism to increase ATP production. One of the mechanisms employed by AMPK to regulate metabolism is protein acetylation. AMPK regulates protein acetylation by at least five distinct mechanisms. First, AMPK phosphorylates and inhibits acetyl-CoA carboxylase (ACC) and thus regulates acetyl-CoA homeostasis. Since acetyl-CoA is a substrate for all lysine acetyltransferases (KATs), AMPK affects the activity of KATs by regulating the cellular level of acetyl-CoA. Second, AMPK activates histone deacetylases (HDACs) sirtuins by increasing the cellular concentration of NAD+, a cofactor of sirtuins. Third, AMPK inhibits class I and II HDACs by upregulating hepatic synthesis of α-hydroxybutyrate, a natural inhibitor of HDACs. Fourth, AMPK induces translocation of HDACs 4 and 5 from the nucleus to the cytoplasm and thus increases histone acetylation in the nucleus. Fifth, AMPK directly phosphorylates and downregulates p300 KAT. On the other hand, protein acetylation regulates AMPK activity. Sirtuin SIRT1-mediated deacetylation of liver kinase B1 (LKB1), an upstream kinase of AMPK, activates LKB1 and AMPK. AMPK phosphorylates and inactivates ACC, thus increasing acetyl-CoA level and promoting LKB1 acetylation and inhibition. In yeast cells, acetylation of Sip2p, one of the regulatory β-subunits of the SNF1 complex, results in inhibition of SNF1. This results in activation of ACC and reduced cellular level of acetyl-CoA, which promotes deacetylation of Sip2p and activation of SNF1. Thus, in both yeast and mammalian cells, AMPK/SNF1 regulate protein acetylation and are themselves regulated by protein acetylation.

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Carbonic anhydrase and soluble adenylate cyclase regulation of cystic fibrosis cellular phenotypes

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00022.2021 ◽

2022 ◽

Author(s):

Kathleen Boyne ◽

Deborah A. Corey ◽

Pan Zhao ◽

Binyu Lu ◽

Walter F Boron ◽

...

Keyword(s):

Cystic Fibrosis ◽

Epithelial Cells ◽

Carbonic Anhydrase ◽

Adenylate Cyclase ◽

Protein Expression ◽

Cell Biology ◽

Intracellular Transport ◽

Human Nasal Epithelial Cells ◽

Primary Mouse ◽

Cellular Phenotypes

Several aspects of the cell biology of cystic fibrosis (CF) epithelial cells are altered including impaired lipid regulation, disrupted intracellular transport, and impaired microtubule regulation. It is unclear how the loss of cystic fibrosis transmembrane conductance regulator (CFTR) function leads to these differences. It is hypothesized that the loss of CFTR function leads to altered regulation of carbonic anhydrase (CA) activity resulting in cellular phenotypic changes. In this study, it is demonstrated that CA2 protein expression is reduced in CF model cells, primary mouse nasal epithelial (MNE) cells, excised MNE tissue, and primary human nasal epithelial cells (p<0.05). This corresponds to a decrease in CA2 RNA expression measured by qPCR as well as an overall reduction in CA activity in primary CF MNEs. The addition of CFTR-inhibitor-172 to WT MNE cells for ≥24 h mimics the significantly lower protein expression of CA2 in CF cells. Treatment of CF cells with L-Phenylalanine (L-Phe), an activator of CA activity, restores endosomal transport through an effect on microtubule regulation in a manner dependent on soluble adenylate cyclase (sAC). This effect can be blocked with the CA2-selective inhibitor dorzolamide. These data suggest the loss of CFTR function leads to the decreased expression of CA2 resulting in the downstream cell signaling alterations observed in CF.

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