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

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.

scholarly journals MAPK signaling pathways regulate IL-8 mRNA stability and IL-8 protein expression in cystic fibrosis lung epithelial cell lines

2011 ◽  
Vol 300 (1) ◽  
pp. L81-L87 ◽  
Author(s):  
Sharmistha Bhattacharyya ◽  
Usha Gutti ◽  
Jose Mercado ◽  
Chad Moore ◽  
Harvey B. Pollard ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Protein Expression ◽  
Signaling Pathways ◽  
Mrna Stability ◽  
Mapk Signaling ◽  
Lung Epithelial Cells ◽  
Lung Epithelial ◽  
The Stability ◽  
Mapk Signaling Pathways

Cystic fibrosis (CF) is characterized by a massive proinflammatory phenotype in the lung, caused by mutations in the CFTR gene. IL-8 and other proinflammatory mediators are elevated in the CF airway, and the immediate mechanism may depend on disease-specific stabilization of IL-8 mRNA in CF lung epithelial cells. MAPK signaling pathways impact directly on IL-8 protein expression in CF cells, and we have hypothesized that the mechanism may also involve stabilization of the IL-8 mRNA. To test this hypothesis, we have examined the effects of pharmacological and molecular inhibitors of p38, and downstream MK2, ERK1/2, and JNK, on stability of IL-8 mRNA in CF lung epithelial cells. We previously showed that tristetraprolin (TTP) was constitutively low in CF and that raising TTP destabilized the IL-8 mRNA. We therefore also tested these effects on CF lung epithelial cells stably expressing TTP. TTP binds to AU-rich elements in the 3′-UTR of the IL-8 mRNA. We find that inhibition of p38 and ERK1/2 reduces the stability of IL-8 mRNA in parental CF cells. However, neither intervention further lowers TTP-dependent destabilization of IL-8 mRNA. By contrast, inhibition of the JNK-2 pathway has no effect on IL-8 mRNA stability in parental CF cell, but rather increases the stability of the message in cells expressing high levels of TTP. However, we find that inhibition of ERK1/2 or p38 leads to suppression of the effect of JNK-2 inhibition on IL-8 mRNA stability. These data thus lend support to our hypothesis that constitutive MAPK signaling and proteasomal activity might also contribute, along with aberrantly lower TTP, to the proinflammatory phenotype in CF lung epithelial cells by increasing IL-8 mRNA stability and IL-8 protein expression.

scholarly journals Dimeric cystic fibrosis transmembrane conductance regulator exists in the plasma membrane

2003 ◽  
Vol 374 (3) ◽  
pp. 793-797 ◽  
Author(s):  
Mohabir RAMJEESINGH ◽  
Jackie F. KIDD ◽  
Ling Jun HUAN ◽  
Yanchun WANG ◽  
Christine E. BEAR
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Cell Biology ◽  
Plasma Membranes ◽  
Chinese Hamster Ovary Cells ◽  
Apical Surface ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
The Impact ◽  
Cystic Fibrosis Transmembrane

CFTR (cystic fibrosis transmembrane conductance regulator) mediates chloride conduction across the apical membrane of epithelia, and mutations in CFTR lead to defective epithelial fluid transport. Recently, there has been considerable interest in determining the quaternary structure of CFTR at the cell surface, as such information is a key to understand the molecular basis for pathogenesis in patients harbouring disease-causing mutations. In our previous work [Ramjeesingh, Li, Kogan, Wang, Huan and Bear (2001) Biochemistry 40, 10700–10706], we showed that monomeric CFTR is the minimal functional form of the protein, yet when expressed in Sf 9 cells using the baculovirus system, it also exists as dimers. The purpose of the present study was to determine if dimeric CFTR exists at the surface of mammalian cells, and particularly in epithelial cells. CFTR solubilized from membranes prepared from Chinese-hamster ovary cells stably expressing CFTR and from T84 epithelial cells migrates as predicted for monomeric, dimeric and larger complexes when subjected to sizing by gel filtration and analysis by non-dissociative electrophoresis. Purification of plasma membranes led to the enrichment of CFTR dimers and this structure exists as the complex glycosylated form of the protein, supporting the concept that dimeric CFTR is physiologically relevant. Consistent with its localization in plasma membranes, dimeric CFTR was labelled by surface biotinylation. Furthermore, dimeric CFTR was captured at the apical surface of intact epithelial cells by application of a membrane-impermeable chemical cross-linker. Therefore it follows from the present study that CFTR dimers exist at the surface of epithelial cells. Further studies are necessary to understand the impact of dimerization on the cell biology of wild-type and mutant CFTR proteins.

scholarly journals Ibuprofen regulation of microtubule dynamics in cystic fibrosis epithelial cells

2016 ◽  
Vol 311 (2) ◽  
pp. L317-L327 ◽  
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.

Macrolide Treatment Decreased the Size of Nasal Polyps and IL-8 Levels in Nasal Lavage

2000 ◽  
Vol 14 (3) ◽  
pp. 143-148 ◽  
Author(s):  
Takechiyo Yamada ◽  
Shigeharu Fujieda ◽  
Shigehito Mori ◽  
Hideyuki Yamamoto ◽  
Hitoshi Saito
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Chronic Rhinosinusitis ◽  
Nasal Polyps ◽  
Experimental Studies ◽  
Nasal Lavage ◽  
Nasal Epithelial Cells ◽  
Diffuse Panbronchiolitis ◽  
Human Nasal Epithelial Cells

Recently, epidemiologic and experimental studies have been reported that long-term macrolides are effective for the treatment of chronic airway inflammatory diseases including diffuse panbronchiolitis, chronic rhinosinusitis, and cystic fibrosis (Jaffe A, Francis J, Rosenthal M, et al. Long-term azithromycin may improve lung function in children with cystic fibrosis. Lancet 351:420, 1998), and that macrolides can directly reduce the production of IL-8 by nasal epithelial cells (Suzuki H, Shimomura A, Ikeda K, et al. Inhibitory effect of macrolides on interleukin-8 secretion from cultured human nasal epithelial cells. Laryngoscope 107:1661–1666, 1997). In this study we administered macrolides with 14-membered rings to patients with nasal polyps due to chronic rhinosinusitis for at least 3 months and measured the IL-8 level in nasal lavage from those patients. The IL-8 levels in nasal lavage from patients with nasal polyps were reduced during macrolide treatment. There was significant correlation between decreased IL-8 levels in nasal lavage and the clinical effect of macrolides on the size of the nasal polyps. In the group whose polyps were reduced in size, the IL-8 levels dramatically decreased from 231.2 pg/mL to 44.0 pg/mL (p < 0.05), and were significantly higher before macrolide treatment than those in the group whose polyps showed no change (p < 0.005). This reduction in IL-8 may be an important aspect of the effect of macrolide treatment on nasal polyps in chronic rhinosinusitis.

Extent of Rescue of F508del-CFTR Function by VX-809 and VX-770 in Human Nasal Epithelial Cells Correlates with SNP rs7512462 in SLC26A9 Gene in F508del/F508del Cystic Fibrosis Patients

2018 ◽  
Author(s):  
Arthur Kmit ◽  
Fernando Augusto de Lima Marson ◽  
Stéphanie Villa-Nova Pereira ◽  
Adriana Mendes Vinagre ◽  
Gabriela Silva Leite ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Nasal Epithelial Cells ◽  
Human Nasal Epithelial Cells

Effects of various secretagogues on [Ca2+]i in cultured human nasal epithelial cells

1991 ◽  
Vol 69 (8) ◽  
pp. 1211-1216 ◽  
Author(s):  
Robert A. Harris ◽  
Kenneth G. Baimbridge ◽  
Michael A. Bridges ◽  
John E. Phillips
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Prostaglandin E ◽  
Epithelial Tissue ◽  
Nasal Epithelial Cells ◽  
Fura 2 ◽  
Culture Cells ◽  
Human Nasal Epithelial Cells ◽  
Messenger System ◽  
Electrolyte Secretion

Recent studies conducted on cultured canine tracheal cells have suggested that a complex relationship exists between cAMP and Ca2+ in the control of electrolyte secretion. The goal of this study was to determine if the Ca2+ second messenger system functions in a similar fashion in cultured human nasal epithelial cells, a tissue in which control of electrolyte secretion is known to be disrupted in the genetic disease, cystic fibrosis. Human nasal epithelial tissue was obtained as a by-product of surgery and put into monolayer cell culture. After 4–5 days in culture, cells were loaded with fura-2 and intracellular free Ca2+ measured as previously reported. We found that bradykinin increased intracellular free Ca2+ in all cells tested, whereas isoproterenol increased intracellular free Ca2+ in only half the cells tested, suggesting that more than one transporting cell type may be present in this tissue. Epinephrine and prostaglandin E2 had no effect on intracellular free Ca2+. We found that the voltage-sensitive Ca2+ channel blocker verapamil had no effect on the bradykinin-induced change in intracellular free Ca2+. Removal of Ca2+ from the bathing saline only slightly attenuated the increase in intracellular free Ca2+ that resulted from stimulation with bradykinin. We conclude that the source of the increased intracellular free CA2+, observed during stimulation with secretagogues, was primarily intracellular stores.Key words: airway epithelia, [Ca2+]i, calcium, bradykinin, cystic fibrosis, fura-2.

scholarly journals Extent of rescue of F508del-CFTR function by VX-809 and VX-770 in human nasal epithelial cells correlates with SNP rs7512462 in SLC26A9 gene in F508del/F508del Cystic Fibrosis patients

2019 ◽  
Vol 1865 (6) ◽  
pp. 1323-1331 ◽  
Author(s):  
Arthur Kmit ◽  
Fernando Augusto Lima Marson ◽  
Stéphanie Villa-Nova Pereira ◽  
Adriana Mendes Vinagre ◽  
Gabriela Silva Leite ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Nasal Epithelial Cells ◽  
Human Nasal Epithelial Cells

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

2019 ◽  
Vol 316 (6) ◽  
pp. L1081-L1093
Author(s):  
Sharon M. Rymut ◽  
Binyu Lu ◽  
Aura Perez ◽  
Deborah A. Corey ◽  
Kata Lamb ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Adverse Events ◽  
Intracellular Transport ◽  
Adenosine Monophosphate ◽  
Cellular Level ◽  
Microtubule Dynamics ◽  
High Dose ◽  
Acetyl Coa Carboxylase ◽  
Acetyl Coa

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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