Serum and glucocorticoid-inducible kinase1 increases plasma membrane wt-CFTR in human airway epithelial cells by inhibiting its endocytic retrieval

Background: Chloride (Cl) secretion by the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) located in the apical membrane of respiratory epithelial cells plays a critical role in maintenance of the airway surface liquid and mucociliary clearance of pathogens. Previously, we and others have shown that the serum and glucocorticoid-inducible kinase-1 (SGK1) increases wild type CFTR (wt-CFTR) mediated Cl transport in Xenopus oocytes by increasing the amount of wt-CFTR protein in the plasma membrane. However, the effect of SGK1 on the membrane abundance of wt-CFTR in airway epithelial cells has not been examined, and the mechanism whereby SGK1 increases membrane wt-CFTR has also not been examined. Thus, the goal of this study was to elucidate the mechanism whereby SGK1 regulates the membrane abundance of wt-CFTR in human airway epithelial cells. Methods and Results: We report that elevated levels of SGK1, induced by dexamethasone, increase plasma membrane abundance of wt-CFTR. Reduction of SGK1 expression by siRNA (siSGK1) and inhibition of SGK1 activity by the SGK inhibitor GSK 650394 abrogated the ability of dexamethasone to increase plasma membrane wt-CFTR. Overexpression of a constitutively active SGK1 (SGK1-S422D) increased plasma membrane abundance of wt-CFTR. To understand the mechanism whereby SGK1 increased plasma membrane wt-CFTR, we examined the effects of siSGK1 and SGK1-S442D on the endocytic retrieval of wt-CFTR. While siSGK1 increased wt-CFTR endocytosis, SGK1-S442D inhibited CFTR endocytosis. Neither siSGK1 nor SGK1-S442D altered the recycling of endocytosed wt-CFTR back to the plasma membrane. By contrast, SGK1 increased the endocytosis of the epidermal growth factor receptor (EGFR). Conclusion: This study demonstrates for the first time that SGK1 selectively increases wt-CFTR in the plasma membrane of human airway epithelia cells by inhibiting its endocytic retrieval from the membrane. © 2014 Bomberger et al

Monomethylarsonous Acid (MMAIII) Has an Adverse Effect on the Innate Immune Response of Human Bronchial Epithelial Cells to Pseudomonas Aeruginosa

Arsenic is the number one contaminant of concern with regard to human health according to the World Health Organization. Epidemiological studies on Asian and South American populations have linked arsenic exposure with an increased incidence of lung disease, including pneumonia, and chronic obstructive pulmonary disease, both of which are associated with bacterial infection. However, little is known about the effects of low dose arsenic exposure, or the contributions of organic arsenic to the innate immune response to bacterial infection. This study examined the effects on Pseudomonas aeruginosa (P. aeruginosa) induced cytokine secretion by human bronchial epithelial cells (HBEC) by inorganic sodium arsenite (iAsIII) and two major metabolites, monomethylarsonous acid (MMAIII) and dimethylarsenic acid (DMAV), at concentrations relevant to the U.S. population. Neither iAsIII nor DMAV altered P. aeruginosa induced cytokine secretion. By contrast, MMAIII increased P. aeruginosa induced secretion of IL-8, IL-6 and CXCL2. A combination of iAsIII, MMAIII and DMAV (10 pbb total) reduced IL-8 and CXCL1 secretion. These data demonstrate for the first time that exposure to MMAIII alone, and a combination of iAsIII, MMAIII and DMAV at levels relevant to the U.S. may have negative effects on the innate immune response of human bronchial epithelial cells to P. aeruginosa

Predicting the seismic behaviour of the foundations of the Messina Strait Bridge

This paper presents some of the geotechnical studies carried out for the seismic design of the one-span suspension bridge across the Messina Strait, that is to connect Sicily with mainland Italy. These studies included advanced geotechnical characterisation, through in situ and laboratory tests, estimate of site stability involving both liquefaction analysis and submerged slope stability, evaluation of soil-foundation stiffness for spectral analysis of the superstructure, 3D FE static calculations, evaluation of anchor block performance under seismic conditions, and full dynamic analyses of the soil-structure interaction. The paper summarises the main results obtained from the geotechnical characterisation of the foundation soils, reports the approach adopted for evaluating the seismic performance of the anchor blocks through a modified Newmark-type calculation, and presents the study of the soil-structure interaction carried out through a series of two-dimensional, plane strain numerical analyses. In these analyses, in addition to the embedded foundation elements, the models included a simplified structural description of the bridge towers specifically designed to reproduce their first vibrations modes, that were deemed to have the most significant influence on the system's dynamic response. The illustration is limited to the foundation systems of the bridge located on the Sicily shore. © 2013 Springer Science+Business Media Dordrecht

SGK1 inhibits wt-CFTR endocytosis.

<p>(A) and (B), siRNA-mediated knockdown of SGK1 in cells treated with dexamethasone (50 nM for 4 hours) enhanced the endocytic removal of wt-CFTR from the apical membrane. CFBE cells were transfected with siSGK1 or a scrambled negative control (siNeg) and 24 hours later treated with dexamethasone (50 nM) for 4 hours. (A) Representative western blot and (B) summary of data. Wt-CFTR endocytosis was measured by a method described previously in detail and in Methods <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089599#pone.0089599-Bomberger2" target="_blank">[12]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089599#pone.0089599-Bomberger3" target="_blank">[22]</a>. In brief, in three sets of cells apical membrane proteins were biotinylated at 4°C using EZ-Link™ Sulfo-NHS-SS-Biotin. Subsequently, one set of cells (lane 1) were then lysed, and the biotinylated proteins were isolated by streptavidin-agarose beads, eluted into SDS sample buffer, separated by 7.5% SDS-PAGE, and probed for wt-CFTR and ezrin. Thus, lane 1 represents the amount of wt-CFTR in the apical membrane at time = 0. A second set of cells (lane 3) were also biotinylated at 4°C and then warmed to 37°C for 5 min to allow biotinylated wt-CFTR to be endocytosed. Subsequently, the cells were cooled to 4°C, and the disulfide bonds on Sulfo-NHS-SS-biotinylated proteins remaining in the apical membrane were reduced by GSH added to the apical solution for a total of 90 min at 4°C. The cells were then lysed, and the biotinylated proteins were isolated by streptavidin-agarose beads, eluted into SDS sample buffer, separated by 7.5% SDS-PAGE, and probed for wt-CFTR and ezrin. Thus, lane 3 represent the amount of wt-CFTR in the apical membrane at time = 0 that was endocytosed in 5 minutes. Lane 2 demonstrates that GSH reduced the disulfide bonds on Sulfo-NHS-SS-biotinylated proteins in the apical membrane. Cells in this lane were biotinylated at 4°C and the disulfide bonds on Sulfo-NHS-SS-biotinylated proteins remaining in the apical membrane were reduced by GSH added to the apical solution for a total of 90 min at 4°C. The amount of biotinylated wt-CFTR remaining in the plasma membrane after GSH treatment at 4°C and without the 37°C warming was considered background (<5% compared with the amount of biotinylated wt-CFTR at 4°C without GSH treatment, i.e., lane 1) and was subtracted from the wt-CFTR biotinylated after warming to 37°C at each time point. wt-CFTR endocytosis is reported as the amount of CFTR in lane 3 (minus background) divided by the amount in lane 1 (minus background) X 100. Quantification of three experiments. *p<0.05 versus siNeg (control). (C) and (D), Constitutively active SGK (SGK1-S422D) reduced the endocytic rate of wt-CFTR from the apical membrane compared to a dominant negative SGK1 (K127N). CFBE cells were transfected with SGK1-S422D or SGK1-K127N and wt-CFTR endocytosis was measured as described above. Representative western blot (C), and summary of the data (D). Quantification of three-six experiments. *p<0.05 versus K127N.</p

SGK1 did not alter the recycling of endocytosed wt-CFTR from endosomes back to the apical membrane.

<p>siRNA-mediated knockdown of SGK1. (A) Representative western blot, and (B) summary of the data. The recycling assay has been described in detail previously and in Methods <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089599#pone.0089599-Bomberger2" target="_blank">[12]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089599#pone.0089599-Bomberger3" target="_blank">[22]</a>. In brief, the first three lanes in A are similar to those described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089599#pone-0089599-g006" target="_blank">Figure 6</a> for the endocytosis experiments. Thus, lane 1 represents the amount of wt-CFTR in the plasma membrane at time = 0, lane 3 represents the amount of wt-CFTR endocytosed in 5 minutes and lane 2 shows that GSH cleaves the disulfide bonds on Sulfo-NHS-SS-biotinylated wt-CFTR in the apical membrane. In lane 4 cells were warmed to 37°C for 5 min after biotinylation to load endocytic vesicles with biotinylated proteins. Cells were then cooled to 4°C, and the disulfide bonds on Sulfo-NHS-SS-Biotin-labeled proteins remaining in the plasma membrane were reduced by GSH at 4°C. Subsequently, cells were warmed again to 37°C for 5 min to allow endocytosed and biotinylated wt-CFTR to recycle to the plasma membrane. Cells were then cooled again to 4°C, and the disulfide bonds on biotinylated proteins in the apical membrane were reduced with GSH. The amount of recycled wt-CFTR was calculated as the difference between the amount of biotinylated wt-CFTR after the first and second GSH treatments (minus background)×100. Experiments repeated four-five times. NS, not significant. (<u>C) and (D),</u> The constitutively active SGK1 (SGK1-S422D) does not alter the recycling of wt-CFTR to the apical membrane. (C) Representative western blot, and (D) Summary of the data. The recycling assay was performed as described above. Experiments repeated four-five times. NS, not significant.</p

Dexamethasone increased wt-CFTR abundance in cell lysates (A), and in the apical membrane (B).

<p>Representative western blots and data summaries are presented. (A) Cells were treated with dexamethasone (50 nM) for the indicated times at 37°C and western blot analysis was conducted to measure wt-CFTR and ezrin in cell lysates. The molecular mass of wt-CFTR is ∼180 kDa. Ezrin was monitored as a loading control. n = 7 per time point. *p<0.05 versus 0 time point. (B) Cells were treated with dexamethasone (50 nM) for the indicated time points at 37°C, and subsequently apical plasma membrane proteins were biotinylated using a technique previously described in detail by our laboratory <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089599#pone.0089599-Bomberger2" target="_blank">[12]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089599#pone.0089599-Bomberger3" target="_blank">[22]</a>. Briefly, apical membrane proteins were biotinylated at 4°C using EZ-Link™ Sulfo-NHS-SS-Biotin. Subsequently, cells were lysed, and the biotinylated proteins were isolated by streptavidin-agarose beads, eluted into SDS sample buffer, and separated by 7.5% SDS-PAGE. The blots were probed for wt-CFTR (∼180 kDa) and for ezrin, a cytoplasmic protein that was not present in the biotinylated samples, confirming that biotin is impermeable to cell membranes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089599#pone.0089599-Bomberger2" target="_blank">[12]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089599#pone.0089599-Bomberger3" target="_blank">[22]</a>, and that the procedure to determine apical membrane wt-CFTR does not detect intracellular wt-CFTR. In this and all subsequent biotinylation experiments ezrin was never detected in the biotinylated samples by western blot, demonstrating that only plasma membrane proteins were biotinylated. *p<0.05 versus 0 time point. **p<0.01 versus 0 time point.</p

siSGK1 increased the amount of wt-CFTR in early endosomes, but not in recycling endosomes.

<p>Co-immunoprecipitation studies were conducted to determine the subcellular location of wt-CFTR in siNeg and siSGK1 transfected cells treated with dexamethasone (50 nM for 4 hours). EEA and Rab5a are markers of early endosomes, and Rab11a is a marker of recycling endosomes. wt-CFTR was immunoprecipitated, and co-immunoprecipitated proteins were eluted into SDS sample buffer, and separated by 7.5% SDS-PAGE. The blots were then probed for wt-CFTR, EEA, Rab5a, and Rab11. Co-immunoprecipitation of wt-CFTR with EEA1 and Rab5a identifies the amount of wt-CFTR in early endosomes, and co-immunoprecipitation of wt-CFTR with Rab11a identifies the amount of wt-CFTR in recycling endosomes. Quantification of data for Rab and EEA1 immunoprecipitation with wt-CFTR in siNeg and siSGK1 cells is normalized for the total amount of wt-CFTR immunoprecipitated. Blots in siNeg and siSGK1 experiments were cut for presentation, but were run one the same blot to allow for comparison. Lysate, the amount of EEA1, Rab5a Rab11 and wt-CFTR in cell lysates. CFTR IP, indicates IP with the anti-CFTR antibody and then western blot with the indicated antibody. IgG, immunoprecipitation with a non-specific antibody. (A) Representative western blots and (B) Summary of the data. Experiments repeated three times. *P<0.05 versus siNeg.</p

The SGK inhibitor GSK 650394 blocked the dexamethasone stimulated increase in apical membrane wt-CFTR.

<p>(A) Representative western blot, and (B) Data quantitation. CFBE cells were treated with either vehicle, GSK 650394 (100 nM), an inhibitor of SGK1, dexamethasone (50 nM), or dexamethasone (50 nM), and GSK 650394 (100 nM) for 24 hours at 37°C. The molecular mass of apical plasma membrane wt-CFTR is ∼180 kDa. Experiments performed four times. *p<0.05 versus vehicle. **p<0.05 versus dexamethasone.</p

SGK1 stimulates EGFR endocytosis.

<p>(A) and (B), Constitutively active SGK1 (S422D) increased the endocytic rate of EGFR from the apical membrane compared to a dominant negative SGK1 (K127N). CFBE cells were transfected with SGK1-S422D or SGK1-K127N and EGFR endocytosis was measured as described in detail in Methods and in Figure legends above. Representative western blot (A), and summary of the data (B). Quantification of three experiments. *P<0.05 versus K127N.</p