Estradiol Increases Mucus Synthesis in Bronchial Epithelial Cells

<div><p>Airway epithelial mucus hypersecretion and mucus plugging are prominent pathologic features of chronic inflammatory conditions of the airway (e.g. asthma and cystic fibrosis) and in most of these conditions, women have worse prognosis compared with male patients. We thus investigated the effects of estradiol on mucus expression in primary normal human bronchial epithelial cells from female donors grown at an air liquid interface (ALI). Treatment with estradiol in physiological ranges for 2 weeks caused a concentration-dependent increase in the number of PAS-positive cells (confirmed to be goblet cells by MUC5AC immunostaining) in ALI cultures, and this action was attenuated by estrogen receptor beta (ER-β) antagonist. Protein microarray data showed that nuclear factor of activated T-cell (NFAT) in the nuclear fraction of NHBE cells was increased with estradiol treatment. Estradiol increased NFATc1 mRNA and protein in ALI cultures. In a human airway epithelial (1HAE₀) cell line, NFATc1 was required for the regulation of MUC5AC mRNA and protein. Estradiol also induced post-translational modification of mucins by increasing total fucose residues and fucosyltransferase (FUT-4, -5, -6) mRNA expression. Together, these data indicate a novel mechanism by which estradiol increases mucus synthesis in the human bronchial epithelium.</p></div

Effect of estradiol on Periodic Acid Schiff (PAS)-positive cell staining in air liquid interface (ALI) cultures.

<p>A) ALI cultures (N = 4) were treated with estradiol (vehicle control, 10⁻⁹, 10⁻⁸, 10⁻⁷ M) and ER-α (10⁻⁶ M MPP) or ER-β (10⁻⁶ M PHTPP) antagonists for 2 weeks. Images are representative of 4 different donors with 3 biological replicates (5 µm sections; scale bars = 50 µm). B) Total cell count normalized to millimeters of epithelium by manual counting of nuclei counterstained with hematoxylin (N = 4). C) Quantification of the percentage of PAS stain of the entire cross section of the epithelium (**P<0.01, ****P<0.0001 against control, #P<0.05 against 10⁻⁷ M estradiol-treated group). D) Quantification of cell count for PAS-positive cells and ciliated cells in the apical compartment of the epithelium with increasing estradiol concentration, and in the presence of 10⁻⁶ M PHTPP or MPP with a single fixed concentration of estradiol at 10⁻⁷ M (N = 4). * P<0.05, ## P<0.01 and ****/#### P<0.0001 compared against vehicle control, +++ P<0.001 and ++++ P<0.0001 compared against 10⁻⁷ M estradiol. One-way ANOVA with Bonferroni's test was used in all analyses.</p

Effects of estradiol on fucosyltransferase mRNA expression in ALI cultures.

<p>10⁻⁷ M estradiol (black bars) increased FUT-4, -5 and -6 mRNA expression compared with vehicle control (white bars). Data are expressed as A) absolute change in C_t value normalized to HPRT1 and B) fold increase in mRNA expression over vehicle control normalized using HPRT1. Values shown are mean ± SEM of 4 different donors performed in 3 biological replicates. Total fucose sugar residues in the cytoplasmic protein fractions of ALI cultures were determined by lectin binding assays. Fucose residues were significantly increased with 10⁻⁷ M estradiol using fucose binding lectins, C-D) AAA, E-F) LTA and G-H) UEA-1. D, F, and H are densitometric quantifications of C, E, and G, respectively. The intensity of all bands in each lane in detecting total fucose residues were quantified using the software program Image J and normalized to total protein loaded per lane in milligrams. Data is expressed as fold increase over vehicle control. Values shown are mean ± SEM of experiments performed with N = 4 donors. * P<0.05, ** P<0.01 compared against vehicle control. Non-parametric t-tests were used in all statistical analyses.</p

Effect of estradiol and NFATc1-specific siRNA on NFATc1 and MUC5AC protein expression in 1HAE₀ cells by western blot.

<p>A-B) 24 h 10⁻⁷ M estradiol treatment alone (E2) and 48 h post-transfection with scrambled siRNA (siCon+E2) increases total cellular NFATc1 protein expression (normalised to HSP90). The effect of E2 is attenuated in cells transfected with siRNA against NFATc1 (siNFATc1+E2). C-D) Total cellular MUC5AC protein/HSP90 expression was increased by 10⁻⁷ M estradiol alone (E2) and in scrambled siRNA controls (siCon+E2), but was attenuated by transfection with NFATc1 siRNA (siNFATc1+E2) treatment. Values shown are mean ± SEM of experiments performed in 3 independent experiments. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001 using one-way ANOVA with Bonferroni's test in all analyses.</p

Effect of estradiol and NFATc1-specific siRNA on NFATc1 and MUC5AC mRNA expression in 1HAE₀ cells.

<p>A) Quantitative real-time PCR demonstrates 10⁻⁷ M estradiol stimulates a time-dependent increase in NFATc1 mRNA/HPRT1 expression with maximal increase at 4 h. B) 10⁻⁷ M estradiol stimulates a time-dependent increase in MUC5AC mRNA/HPRT1 expression with maximal increase at 24 h. C) 24 h 10⁻⁷ M estradiol treatment increases NFATc1 mRNA/HPRT1 expression in cells 48 h post-transfection with scrambled control siRNA (siCon+E2) and this effect is reduced by transfection with NFATc1 siRNA (siNFATc1+E2). D) MUC5AC mRNA/HPRT1 expression was increased by 24 h treatment with 10⁻⁷ M estradiol (E2) with scrambled siRNA transfection (siCon+E2), but was attenuated by transfection with NFATc1 siRNA (siNFATc1+E2). E) NFATc1–c4 mRNA expression in cells treated with 100 nM NFATc1 siRNA for 48 h. F) A representative western blot of NFATc1 protein knockdown expression with 100 nM NFATc1 siRNA treatment for 48 h and quantified by densitometry in G) with N = 3. Values shown are mean ± SEM of experiments performed in 3 independent experiments. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001 using one-way ANOVA with Bonferroni's test in A-D and non-parametric t-test was used in E and G.</p

Effects of estradiol on MUC5AC mRNA and protein expression.

<p>A) MUC5AC immunostaining in ALI cultures treated with 10⁻⁷ M estradiol for 2 weeks (goblet cells indicated by red arrows and counterstained with hematoxylin of nuclei). B) Quantification of the number of goblet cells and ciliated cells in the apical compartment of estradiol-treated ALI cultures expressed as a percentage of all cells in the apical compartment. #### P<0.0001 (ciliated cells) and **** P<0.0001 (goblet cells) vs. vehicle control, N = 4. C) MUC5AC mRNA expression normalized to HPRT1 housekeeping using real time PCR. D) MUC5AC protein present in ALI culture secretion was analysed by western blot, normalized by the total volume of apical washes per well and quantified by densitometry of N = 4 donors in duplicate lanes E). All values shown are mean ± SEM of 4 different donors with 3 biological replicates. Non-parametric t-test were used in all analyses.</p

NFAT mRNA and protein expression in 1HAE₀ cells after estradiol stimulation for 24 h.

<p>A) Baseline mRNA expression of NFAT (c1–c4) in 1HAE₀ cells quantified by real-time PCR expressed as absolute change in C_t value normalized to HPRT1. B) NFATc1–c4 mRNA expression normalized to HPRT1 in cells treated with 10⁻⁷ M estradiol for 24 h. Data are expressed as fold increase over vehicle controls. Values shown are mean ± SEM of experiments performed in 3 independent experiments. ****P<0.0001 compared against vehicle control in each gene using non-parametric t-test in B-C.</p

Protein expression of estrogen receptors in excised human large airways of control lungs: A) rabbit immunogobulin-G (IgG) control, B) ER-α and C) ER-β by immunohistochemistry.

<p>Protein expression of estrogen receptors in air liquid interface (ALI) cultures: D) rabbit immunogobulin-G (IgG) control, E) ER-α and F) ER-β. G) Western blot (WB) analysis of ER-α and ER-β protein expression in cytoplasmic and nuclear fraction of control ALI cultures at passage 4. Beta-actin, superoxide dismutase (SOD) and histone 3 were used as house-keeping protein, cytoplasmic and nuclear-specific marker, respectively. Representative WB images of N = 4 in duplicate lanes is shown. H) Quantification of positive cell staining of nuclei in red is expressed as a percentage of total cells on the epithelium of intact human airways and in control ALI cultures. Images are representative of N = 4 female excised airways and N = 4 ALI cultures. Sections were counterstained with hematoxylin for nuclei (blue) and positive stains were indicated by red arrows. (5 µm sections; scale bars = 50 µm). *P<0.05 represents statistical significance using a non-parametric t-test.</p

NFAT mRNA and protein expression in ALI cultures of primary NHBEs after 2 weeks of estradiol stimulation at 10⁻⁷ M.

<p>A) Quantitative real-time PCR demonstrates baseline vehicle control mRNA expression of NFAT (c1–c4) expressed as absolute C_t value normalized to HPRT1. B) NFATc1–c4 mRNA expression normalized to HPRT1 in vehicle vs. 10⁻⁷ M estradiol-treated cultures for 2 weeks. C) Western blot showed total NFATc1 protein expression with HSP90 housekeeping protein in vehicle vs. 10⁻⁷ M estradiol-treated ALI cultures and quantified by densitometry in D) with N = 4. *P<0.05 and **P<0.01 compared against vehicle control using non-parametric t-test and expressed as fold increase over control in B and D.</p

Additional file 1: of Gene expression analysis in asthma using a targeted multiplex array

Supplementary Methods – Methods describing selection of house keeping genes and immunohistochemical staining procedure. Supplementary Tables – Tables containing clinical demographics for subjects, average counts, fold change, and p-value for all genes studied, and all differentially co-expressed genes. Supplementary Figures and Legends – Figures showing sample immunohistochemical staining for proteins of significantly altered genes, co-expression plots. (DOCX 35 kb