Biomedical subjects

J Kelley Bentley

Publications and source records attributed to J Kelley Bentley.

7 recordsLinked to original sources

PubMed2006

Rhinovirus activates interleukin-8 expression via a Src/p110beta phosphatidylinositol 3-kinase/Akt pathway in human airway epithelial cells.

Rhinovirus (RV) is responsible for the majority of common colds and triggers exacerbations of asthma and chronic obstructive lung disease. We have shown that RV serotype 39 (RV39) infection activates phosphatidylinositol 3 (PI 3)-kinase and the serine threonine kinase Akt minutes after infection and that the activation of PI 3-kinase and Akt is required for maximal interleukin-8 (IL-8) expression. Here, we further examine the contributions of Src and PI 3-kinase activation to RV-induced Akt activation and IL-8 expression. Confocal fluorescent microscopy of 16HBE14o- human bronchial epithelial cells showed rapid (10-min) colocalization of RV39 with Src, p85alpha PI 3-kinase, p110beta PI 3-kinase, Akt and Cit-Akt-PH, a fluorescent Akt pleckstrin homology domain which binds PI(3,4,5)P(3). The chemical Src inhibitor PP2 {4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo [3,4-d]pyrimidine} and the PI 3-kinase inhibitor LY294002 each inhibited Akt phosphorylation and the colocalization of RV39 with Akt. Digoxigenin-tagged RV coprecipitated with a Crosstide kinase likely to be Akt, and inhibition of Src blocked kinase activity. Digoxigenin-tagged RV39 colocalized with the lipid raft marker ceramide. In 16HBE14o- and primary mucociliary differentiated human bronchial epithelial cells, inhibition of Src kinase activity with the Src family chemical inhibitor PP2, dominant-negative Src (K297R), and Src small interfering RNA (siRNA) each inhibited RV39-induced IL-8 expression. siRNA against p110beta PI 3-kinase also inhibited IL-8 expression. These data demonstrate that, in the context of RV infection, Src and p110beta PI 3-kinase are upstream activators of Akt and the IL-8 promoter and that RV colocalizes with Src, PI 3-kinase, and Akt in lipid rafts.

Bronchi↗

PubMed2006

Regulation of airway smooth muscle alpha-actin expression by glucocorticoids.

Airway smooth muscle hypertrophy appears to be present in severe asthma. However, the effect of corticosteroids on airway smooth muscle cell size or contractile protein expression has not been studied. We examined the effects of dexamethasone, fluticasone, and salmeterol on contractile protein expression in transforming growth factor (TGF)-beta-treated primary bronchial smooth muscle cells. Dexamethasone and fluticasone, but not salmeterol, each reduced expression of alpha-smooth muscle actin and the short isoform of myosin light chain kinase. Steady-state alpha-actin mRNA level and stability were unchanged, consistent with posttranscriptional control. Fluticasone significantly decreased alpha-actin protein synthesis following treatment with the transcriptional inhibitor actinomycin D, indicative of an inhibitory effect on mRNA translation. Fluticasone also significantly increased alpha-actin protein turnover. Finally, fluticasone reduced TGF-beta-induced incorporation of alpha-actin into filamentous actin, cell length, and cell shortening in response to ACh and KCl. We conclude that glucocorticoids reduce human airway smooth muscle alpha-smooth muscle actin expression and incorporation into contractile filaments, as well as contractile function, in part by attenuation of mRNA translation and enhancement of protein degradation.

Actins↗

PubMed2006

H. influenzae potentiates airway epithelial cell responses to rhinovirus by increasing ICAM-1 and TLR3 expression.

Rhinovirus (RV) is an important trigger of chronic obstructive pulmonary disease (COPD) exacerbations. In addition, respiratory viruses are more likely to be isolated in patients with a history of frequent exacerbations, suggesting that these patients are more susceptible to viral infection. To examine potential mechanisms for cooperative effects between bacterial and viral infection in COPD, we studied the responses of cultured human airway epithelial cells to nontypeable Hemophilus influenzae and RV. In both 16HBE14o- and primary mucociliary-differentiated cells, preincubation with H. influenzae enhanced RV serotype 39-induced protein expression of interleukin (IL)-8, epithelial-derived neutrophil attractant-78, and growth-related oncogene-alpha. H. influenzae infection also increased the binding of RV39 to cultured cells, as well as expression of intercellular adhesion molecule (ICAM)-1 and Toll-like receptor (TLR)-3, receptors for RV and dsRNA, respectively. Neutralizing antibody against tumor necrosis factor-alpha inhibited IL-8 expression induced by H. influenzae and RV39. Finally, siRNA against TLR3 attenuated RV-induced IL-8 expression. We conclude that H. influenzae infection increases airway epithelial cell ICAM-1 and TLR3 expression, leading to enhanced binding of RV and a potentiation of RV-induced chemokine release. These data provide a cellular mechanism by which H. influenzae infection may increase the susceptibility of COPD patients to RV-induced exacerbations.

Cells, Cultured↗

PubMed2005

Transforming growth factor-beta induces airway smooth muscle hypertrophy.

Although smooth muscle hypertrophy is present in asthmatic airways, little is known about the biochemical pathways regulating airway smooth muscle protein synthesis, cell size, or accumulation of contractile apparatus proteins. We sought to develop a model of airway smooth muscle hypertrophy in primary cells using a physiologically relevant stimulus. We hypothesized that transforming growth factor (TGF)-beta induces hypertrophy in primary bronchial smooth muscle cells. Primary human bronchial smooth muscle cells isolated from unacceptable lung donor tissue were studied. Cells were seeded on uncoated plastic dishes at 50% confluence and TGF-beta was added. Experiments were performed in the absence of serum. TGF-beta increased cell size and total protein synthesis, expression of alpha-smooth muscle actin and smooth muscle myosin heavy chain, formation of actomyosin filaments, and cell shortening to acetylcholine. Further, TGF-beta increased airway smooth muscle alpha-actin synthesis in the presence of the transcriptional inhibitor actinomycin D, evidence that translational control is a physiologically important element of the observed hypertrophy. TGF-beta induced the phosphorylation of eukaryotic translation initiation factor-4E-binding protein, a signaling event specifically involved in translational control. Finally, two inhibitors of 4E-binding protein phosphorylation, the phosphoinositol 3-kinase inhibitor LY294002 and a phosphorylation site mutant of 4E-binding protein-1 that dominantly inhibits eukaryotic initiation factor-4E, each blocked TGF-beta-induced alpha-actin expression and cell enlargement. We conclude that TGF-beta induces hypertrophy of primary bronchial smooth muscle cells. Further, phosphorylation of 4E-binding protein is required for the observed hypertrophy.

Acetylcholine↗

PubMed2005

Phosphatidylinositol 3-kinase is required for rhinovirus-induced airway epithelial cell interleukin-8 expression.

Rhinovirus (RV) is a common cause of asthma exacerbations. The signaling mechanisms regulating RV-induced airway epithelial cell responses have not been well studied. We examined the role of phosphatidylinositol (PI) 3-kinase in RV-induced interleukin (IL)-8 expression. Infection of 16HBE14o- human bronchial epithelial cells with RV39 induced rapid activation of PI 3-kinase and phosphorylation of Akt, a downstream effector of PI 3-kinase. RV39 also colocalized with cit-Akt-PH, a citrogen-tagged fluorescent fusion protein encoding the pleckstrin homology domain of Akt, indicating that 3-phosphorylated PI accumulates at the site of RV infection. Inhibition of PI 3-kinase and Akt attenuated RV39-induced NF-kappaB transactivation and IL-8 expression. Inhibition of PI 3-kinase also blocked internalization of labeled RV39 into 16HBE14o- cells, suggesting that the requirement of PI 3-kinase for RV39-induced IL-8 expression, at least in part, relates to its role in viral endocytosis.

Bronchi↗

PubMed2005

4E-binding protein phosphorylation and eukaryotic initiation factor-4E release are required for airway smooth muscle hypertrophy.

The molecular mechanisms of airway smooth muscle hypertrophy, a feature of severe asthma, are poorly understood. We previously established a conditionally immortalized human bronchial smooth muscle cell line with a temperature-sensitive SV40 large T antigen. Temperature shift and loss of large T cause G1-phase cell cycle arrest that is accompanied by increased airway smooth muscle cell size. In the present study, we hypothesized that phosphorylation of eukaryotic initiation factor-4E (eIF4E)-binding protein (4E-BP), which subsequently releases eIF4E and initiates cap-dependent mRNA translation, was required for airway smooth muscle hypertrophy. Treatment of cells with chemical inhibitors of PI 3-kinase and mammalian target of rapamycin blocked protein synthesis and cell growth while decreasing the phosphorylation of 4E-BP and increasing the binding of 4E-BP to eIF4E, consistent with the notion that 4E-BP1 phosphorylation and eIF4E function are required for hypertrophy. To test this directly, we infected cells with a retrovirus encoding a phosphorylation site mutant of 4E-BP1 (AA-4E-BP-1) that dominantly inhibits eIF4E. Upon temperature shift, cells infected with AA-4E-BP-1, but not empty vector, failed to undergo hypertrophic growth. We conclude that phosphorylation of 4E-BP, eIF4E release, and cap-dependent protein synthesis are required for hypertrophy of human airway smooth muscle cells.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Immunoprecipitation of PDE2 phosphorylated and inactivated by an associated protein kinase.

A PDE2A2-associated protein kinase phosphorylates PDE2A2 in vivo and in vitro to inhibit its catalytic activity. Rat brain PDE2A2 may be solubilized using nona (ethylene glycol) mono dodecyl ether (Lubrol 12A9). PDE2A2 exists in a complex with a protein kinase regulating its activity in an adenosine triphosphate-dependent manner. When native or recombinant PDE2 is immunoprecipitated from PC12 cells using an antibody to the amino terminus in a buffer containing Lubrol 12A9, protease inhibitors, and phosphatase inhibitors, a coimmunoprecipitating nerve growth factor-stimulated protein kinase acts to phosphorylate it. PDE2A2 phosphoryla-tion occurs optimally at pH 6.5 in a sodium 2-(4-morpholino)-ethane sulfonate buffer with 5 mM MgCl2 and 1 mM Na3VO4. I describe protocols for producing an antibody to an amino-terminal bacterial fusion protein encoding amino acids 1-251 of PDE2A2 as well as the use of this antibody in immunoprecipitating a PDE2: tyrosine protein-kinase complex from rat brain or PC12 cells.

Animals↗