Sex hormone-dependent regulation of cilia beat frequency in airway epithelium

Abstract

Previous studies have demonstrated a female disadvantage in airway diseases, such as asthma and bronchiectasis. The basis for this sex disparity is unknown. We hypothesized that the female sex hormone, progesterone (P4), inhibits functions of the normal airway mucociliary apparatus. P4 receptor (PR) expression was evaluated in human lung and cultured primary human airway epithelial cells isolated from male and female lung transplant donors. PR expression was restricted to the proximal region of the cilia of airway epithelia, and was similar in men and women. Expression of isoform PR-B was more abundant than PR-A in cells from both sexes. Airway epithelial cell exposure to P4 decreased cilia beat frequency (CBF) by 42.3% (±7.2). Inhibition of CBF was prevented by coadministration of P4 with the active form of estrogen, 17β-estradiol, or the PR antagonist, mifepristone. P4 inhibition was time and dose dependent, with a significant decrease by 8 hours and maximal effect at 24 hours, accompanied by translocation of PR from the cilia to the nucleus. Inhibition of cilia beat was also prevented by treatment of cells with actinomycin D, suggesting that CBF inhibition is a transcriptionally mediated event. Together, these findings indicate that sex hormones influence the function of a key component of the mucociliary apparatus. These mechanisms may contribute to the sex disparity present in airway diseases and provide therapeutic targets for the treatment of these debilitating airway diseases.

Figure 1.

Progesterone (P4) receptor (PR) is expressed in cilia of airway epithelial cells. (A) Lung tissue from lung transplant donors of indicated sex immunostained for PR (red) and cilia marker, acetylated α-tubulin (α-tub; green), overlayed with differential interference contrast microscopy images show airway expression of PR in cilia. (B) Representative high-power images from (A) are shown. Rabbit IgG was substituted for anti-PR antibody in samples shown in the far right panel. Image detail shows PR expression in the proximal cilia (insets, middle panels). Images are representative of at least three samples from each sex. (C) Expression of PR in RNA isolated from human tracheal epithelial cell (hTEC) preparations relative to that in T-47D and SW480 cell lines. Shown are the mean (±SD) of PR in hTEC preparations derived from three different male and female donors, normalized to glyceraldehyde 3-phosphate dehydrogenase. (D) Protein blot analysis for PR-A and -B isoforms in hTECs from four different donors. (E) PR expression in cilia of fully differentiated hTEC preparation (male, air–liquid interface [ALI] Day 70) immunostained as in (A), viewed en face. Inset shows PR expression in hTECs sectioned from cells on membrane. Scale bars, 100 μm (A), 30 μm (B), 10 μm (E).

Figure 2.

PR localization is differentiation and ligand dependent in airway epithelial cells. (A) Differentiation-dependent PR expression of hTEC preparations immunostained for PR (red) and acetylated α-tubulin (green) shown en face. Nuclei are stained with DAPI (blue). ALI Day 3 shows PR in the nuclei of all cells. As differentiation proceeds, PR moves to the cytoplasm in some cells (ALI Day 14). Inset shows hTECs immunostained for PR (red) and Foxj1 (green) during differentiation, demonstrating cytoplasmic PR during early ciliogenesis (ALI Day 11). In differentiated hTECs, PR is expressed in the cilia (ALI Day 22). Top right panel shows PR expression in cilia, and top left panel shows PR expression in nuclei of nonciliated cells, within a plane of focus below the level of the cilia. (B) P4-induced nuclear localization of PR in fully differentiated hTEC preparations. hTECs treated with P4, mifepristone (Mife), or P4 plus mifepristone, and immunostained as in (A). (C) Protein blot analysis of hTECs treated as in (B) show no change in the expression of PR isoforms. (D) Densitometry analysis of studies in (C) expressed as the mean (±SD) of fold change of PR-A and -B normalized to actin and relative to vehicle-treated samples from three independent experiments. Scale bars, 10 μm in (A and B).

Figure 3.

P4 decreased cilia beat frequency (CBF) in hTEC preparations. Baseline CBF was measured at time 0 before treatments. (A) Dose–response of P4 on change in CBF measured after 24 hours of P4 treatment. *P < 0.001 relative to baseline. (B) Time-dependent effect of P4 on CBF relative to pretreatment baseline with recovery after 24-hour washout (WO). *P < 0.001 relative to 0–6 hours and **P < 0.001 relative to 0–12 hours. (C) CBF measured 24 hours after P4 (20 μM) or vehicle of hTECs from female and male donors. *P < 0.001 relative to vehicle conditions. Baseline CBF of male and female samples was not different (P = 0.08). (D and E) Effect of blockade of P4 by Mife after 24 hours. *P < 0.001 relative to all other conditions on absolute and relative CBF. (F) 17β-estradiol (E2) prevents P4 inhibition of CBF after 24 hours. *P < 0.001 relative to all other conditions. Data shown are the mean (±SD). CBF measured at 37°C from 5 to 10 fields of each sample from 3 to 10 male and female donors. (G) Representative images of PR localization after vehicle, P4 treatment, and WO, demonstrating PR trafficking. (H) E2 inhibits P4-mediated PR trafficking. Cells were immunostained as in Figure 2B. Scale bars, 10 μm in (G and H).

Figure 4.

P4-dependent inhibition of CBF is abrogated by actinomycin D (AD). (A) CBF of hTEC preparations treated with vehicle, P4, P4 plus AD, or AD alone at indicated times. *P < 0.001 relative to time 0. (B) Representative protein blot analysis of PR expression after 24 hours, as in Figure 2. Cells were exposed to AD for 24 hours before adding P4, and were then treated for indicated additional times.