Urokinase Receptor mRNA Stability Involves Tyrosine Phosphorylation in Lung Epithelial Cells

Interleukin (IL)-8, a C-X-C chemokine, is a potent chemoattractant and an activator for neutrophils, T cells, and other immune cells. The airway and respiratory epithelia play important roles in the initiation and modulation of inflammatory responses via production of cytokines and surfactant. The association between elevated levels of nitric oxide (NO) and IL-8 in acute lung injury associated with sepsis, acute respiratory distress syndrome, respiratory syncytial virus infection in infants, and other inflammatory diseases suggested that NO may play important roles in the control of IL-8 gene expression in the lung. We investigated the role of NO in the control of IL-8 gene expression in H441 lung epithelial cells. We found that a variety of NO donors significantly induced IL-8 mRNA levels, and the increase in IL-8 mRNA was associated with an increase in IL-8 protein. NO induction of IL-8 mRNA was due to increases in IL-8 gene transcription and mRNA stability. NO induction of IL-8 mRNA levels was not inhibited by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one and KT-5823, inhibitors of soluble guanylate cyclase and protein kinase G, respectively, and 8-bromo-cGMP did not increase IL-8 mRNA levels. This indicated that NO induces IL-8 mRNA levels independently of changes in the intracellular cGMP levels. NO induction of IL-8 mRNA was significantly reduced by inhibitors of extracellular regulated kinase and protein kinase C. IL-8 induction by NO was also reduced by hydroxyl radical scavengers such as dimethyl sulfoxide and dimethylthiourea, indicating the involvement of hydroxyl radicals in the induction process. NO induction of IL-8 gene expression could be a significant contributing factor in the initiation and induction of inflammatory response in the respiratory epithelium.

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Tristetraprolin regulates IL-8 mRNA stability in cystic fibrosis lung epithelial cells

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.90601.2008 ◽

2009 ◽

Vol 296 (6) ◽

pp. L1012-L1018 ◽

Cited By ~ 27

Author(s):

Nagaraja Sethuraman Balakathiresan ◽

Sharmistha Bhattacharyya ◽

Usha Gutti ◽

Robert P. Long ◽

Catherine Jozwik ◽

...

Keyword(s):

Cystic Fibrosis ◽

Epithelial Cells ◽

Mrna Stability ◽

Posttranscriptional Regulation ◽

Lung Epithelial Cells ◽

Cellular Phenotype ◽

Lung Epithelial ◽

Vitro Analysis ◽

In Vitro Analysis

Cystic fibrosis (CF) is due to mutations in the CFTR gene and is characterized by hypersecretion of the proinflammatory chemokine IL-8 into the airway lumen. Consequently, this induces the highly inflammatory cellular phenotype typical of CF. Our initial studies revealed that IL-8 mRNA is relatively stable in CF cells compared with those that had been repaired with [WT]CFTR (wild-type CFTR). Relevantly, the 3′-UTR of IL-8 mRNA contains AU-rich sequences (AREs) that have been shown to mediate posttranscriptional regulation of proinflammatory genes upon binding to ARE-binding proteins including Tristetraprolin (TTP). We therefore hypothesized that very low endogenous levels of TTP in CF cells might be responsible for the relative stability of IL-8 mRNA. As predicted, increased expression of TTP in CF cells resulted in reduced stability of IL-8 mRNA. An in vitro analysis of IL-8 mRNA stability in CF cells also revealed a TTP-induced enhancement of deadenylation causing reduction of IL-8 mRNA stability. We conclude that enhanced stability of IL-8 mRNA in TTP-deficient CF lung epithelial cells serve to drive the proinflammatory cellular phenotype in the CF lung.

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

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00051.2010 ◽

2011 ◽

Vol 300 (1) ◽

pp. L81-L87 ◽

Cited By ~ 32

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.

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The fibrinolytic system and the regulation of lung epithelial cell proteolysis, signaling, and cellular viability

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.90349.2008 ◽

2008 ◽

Vol 295 (6) ◽

pp. L967-L975 ◽

Cited By ~ 46

Author(s):

Sreerama Shetty ◽

Joseph Padijnayayveetil ◽

Torry Tucker ◽

Dorota Stankowska ◽

Steven Idell

Keyword(s):

Epithelial Cell ◽

Epithelial Cells ◽

Plasminogen Activator ◽

Mrna Stability ◽

Fibrinolytic System ◽

Lung Epithelial Cells ◽

Lung Epithelial Cell ◽

Plasminogen Activator Inhibitor 1 ◽

Lung Epithelial ◽

Pai 1

The urokinase-type plasminogen activator (uPA), its receptor (uPAR), and plasminogen activator inhibitor-1 (PAI-1) are key components of the fibrinolytic system and are expressed by lung epithelial cells. uPA, uPAR, and PAI-1 have been strongly implicated in the pathogenesis of acute lung injury (ALI) and pulmonary fibrosis. Recently, it has become clear that regulation of uPA, uPAR, and PAI-1 occurs at the posttranscriptional level of mRNA stability in lung epithelial cells. uPA further mediates its own expression in these cells as well as that of uPAR and PAI-1 through induction of changes in mRNA stability. In addition, uPA-mediated signaling controls the expression of the tumor suppressor protein p53 in lung epithelial cells at the posttranslational level. p53 has recently been shown to be a trans-acting uPA, uPAR, and PAI-1 mRNA-binding protein that regulates the stability of these mRNAs. It is now clear that signaling initiated by uPA mediates dose-dependent regulation of lung epithelial cell apoptosis and likewise involves changes in p53, uPA, uPAR, and PAI-1 expression. These findings demonstrate that the uPA-uPAR-PAI-1 system of lung epithelial cells mediates a broad repertoire of responses that encompass but extend well beyond traditional fibrinolysis, involve newly recognized interactions with p53 that influence the viability of the lung epithelium, and are thereby implicated in the pathogenesis of ALI and its repair.

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Urokinase induces activation of STAT3 in lung epithelial cells

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00476.2005 ◽

2006 ◽

Vol 291 (4) ◽

pp. L772-L780 ◽

Cited By ~ 13

Author(s):

Sreerama Shetty ◽

Gadiparthi N. Rao ◽

Douglas B. Cines ◽

Khalil Bdeir

Keyword(s):

Catalytic Activity ◽

Epithelial Cells ◽

Dna Synthesis ◽

Tyrosine Phosphorylation ◽

Lung Epithelial Cells ◽

Time Dependent ◽

Dependent Manner ◽

Single Chain ◽

Amino Terminal ◽

Lung Epithelial

Urokinase-type plasminogen activator (uPA) is a serine protease that plays a major role in diverse physiological and pathological processes. Studies from our laboratory have shown that exposure of human lung epithelial cells to uPA induces proliferation. To understand uPA mitogenic signaling events, we sought to elucidate its effects on tyrosine phosphorylation in a human bronchial epithelial cell line (Beas2B). uPA induced tyrosine phosphorylation of several proteins in a time-dependent manner. One of these proteins was identified as the 91-kDa signal transduction activator transcription (Stat)3 moiety. Tyrosine phosphorylation of Stat3 by uPA was time dependent. uPA induced Stat3-DNA binding activity in a time-dependent manner. uPA-induced Stat3 activation does not require uPA catalytic activity, as the uPA amino-terminal fragment alone was as potent as active two-chain uPA (tcuPA) in causing this effect. Single-chain uPA likewise induced tyrosine phosphorylation of Stat3 to a similar extent as intact tcuPA. Plasmin did not alter uPA-induced Stat3 activation. Furthermore, transfection of Beas2B cells with dominant-negative Stat3 blocked uPA-induced DNA synthesis. These results reveal for the first time that the uPA-uPAR interaction leads to activation of Stat3, independent of its catalytic activity but dependent on its interaction with its receptor, uPAR, leading to DNA synthesis in lung epithelial cells.

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cAMP stimulates Na+ transport in rat fetal pneumocyte: involvement of a PTK- but not a PKA-dependent pathway

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1999.277.4.l727 ◽

1999 ◽

Vol 277 (4) ◽

pp. L727-L736 ◽

Cited By ~ 9

Author(s):

Naomi Niisato ◽

Yasushi Ito ◽

Yoshinori Marunaka

Keyword(s):

Epithelial Cells ◽

Tyrosine Phosphorylation ◽

Short Circuit ◽

Lung Epithelial Cells ◽

Cell Shrinkage ◽

Inhibitory Peptide ◽

Stimulatory Action ◽

Lung Epithelial ◽

Distal Lung ◽

Dependent Pathway

To study a cAMP-mediated signaling pathway in the regulation of amiloride-sensitive Na+ transport in rat fetal distal lung epithelial cells, we measured an amiloride-sensitive short-circuit current (Na+ transport). Forskolin, which increases the cytosolic cAMP concentration, stimulated the Na+ transport. Forskolin also activated cAMP-dependent protein kinase (PKA). A β-adrenergic agonist and cAMP mimicked the forskolin action. PKA inhibitors KT-5720, H-8, and myristoylated PKA-inhibitory peptide amide-(14—22) did not influence the forskolin action. These results suggest that forskolin stimulates Na+ transport through a PKA-independent pathway. Furthermore, forskolin increased tyrosine phosphorylation of ∼70- to 80-, ∼97-, and ∼110- to 120-kDa proteins. Protein tyrosine kinase (PTK) inhibitors (tyrphostin A23 and genistein) abolished the forskolin action. Moreover, 5-nitro-2-(3-phenylpropylamino)benzoate (a Cl−-channel blocker) prevented the stimulatory action of forskolin on Na+ transport via abolishment of the forskolin-induced cell shrinkage and tyrosine phosphorylation. Based on these results, we conclude that forskolin (and cAMP) stimulates Na+ transport in a PTK-dependent but not a PKA-dependent pathway by causing cell shrinkage, which activates PTK in rat fetal distal lung epithelial cells.

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A Primate-Specific RNA-Binding Protein (RBMXL3) Is Involved in Glucocorticoid Regulation of Human Pulmonary Surfactant Protein B (SP-B) mRNA Stability

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00022.2020 ◽

2021 ◽

Author(s):

Lidan Liu ◽

Xiangli Liu ◽

Weizhen Bi ◽

Joseph L Alcorn

Keyword(s):

Epithelial Cells ◽

Mrna Stability ◽

Pulmonary Surfactant ◽

Rna Binding ◽

Surfactant Protein ◽

Lung Epithelial Cells ◽

Mouse Lung ◽

Surfactant Protein B ◽

Lung Epithelial ◽

Protein B

The ability of pulmonary surfactant to reduce alveolar surface tension requires adequate levels of surfactant protein B (SP-B). Dexamethasone (DEX) increases human SP-B expression, in part, through increased SP-B mRNA stability. A 30 nt-long hairpin element (RBE) in the 3'-untranslated region of human SP-B mRNA mediates both DEX-induced and intrinsic mRNA stability, but the mechanism is unknown. Proteomic analysis of RBE-interacting proteins identified a primate-specific protein; RNA binding motif X-linked-like-3 (RBMXL3). siRNA directed against RBMXL3 reduces DEX-induced SP-B mRNA expression in human bronchoalveolar cells. Human SP-B mRNA stability, measured by our dual cistronic plasmid assay, is unaffected by DEX in mouse lung epithelial cells lacking RBMXL3, but DEX increases human SP-B mRNA stability when RBMXL3 is expressed and requires the RBE. In the absence of DEX, RBE interacts with cellular proteins, reducing intrinsic SP-B mRNA stability in human and mouse lung epithelial cells. RBMXL3 specifically binds the RBE in vitro while RNA immunoprecipitation and affinity chromatography analyses indicate that binding is enhanced in the presence of DEX. These results describe a model where intrinsic stability of human SP-B mRNA is reduced through binding of cellular mRNA decay factors to RBE, which is then relieved through DEX-enhanced binding of primate-specific RBMXL3.

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