17beta-Estradiol inhibits Ca2+-dependent homeostasis of airway surface liquid volume in human cystic fibrosis airway epithelia

Abstract

Normal airways homeostatically regulate the volume of airway surface liquid (ASL) through both cAMP- and Ca2+-dependent regulation of ion and water transport. In cystic fibrosis (CF), a genetic defect causes a lack of cAMP-regulated CFTR activity, leading to diminished Cl- and water secretion from airway epithelial cells and subsequent mucus plugging, which serves as the focus for infections. Females with CF exhibit reduced survival compared with males with CF, although the mechanisms underlying this sex-related disadvantage are unknown. Despite the lack of CFTR, CF airways retain a limited capability to regulate ASL volume, as breathing-induced ATP release activates salvage purinergic pathways that raise intracellular Ca2+ concentration to stimulate an alternate pathway to Cl- secretion. We hypothesized that estrogen might affect this pathway by reducing the ability of airway epithelia to respond appropriately to nucleotides. We found that uridine triphosphate-mediated (UTP-mediated) Cl- secretion was reduced during the periovulatory estrogen maxima in both women with CF and normal, healthy women. Estrogen also inhibited Ca2+ signaling and ASL volume homeostasis in non-CF and CF airway epithelia by attenuating Ca2+ influx. This inhibition of Ca2+ signaling was prevented and even potentiated by estrogen antagonists such as tamoxifen, suggesting that antiestrogens may be beneficial in the treatment of CF lung disease because they increase Cl- secretion in the airways.

Figure 1. UTP-activated Cl^– secretion changes with the menstrual cycle.

Females with and without CF calculated their high– and low–E2 level days based on the onset of menses, and nasal PDs were recorded at these times. White bars represent low–E2 level days and black bars represent high–E2 level days. (A and B) Typical traces showing sequential addition of amiloride, a low Cl^– solution, and UTP on low and high days respectively. T, time. (C) Paired mean basal nasal PDs measured on low– and high–E2 level days (n = 12). (D) Paired mean changes in nasal PD in normal subjects on low- and high-level E2 days following amiloride addition and following perfusion of a low Cl^– solution and a low Cl^– solution containing 100 μM UTP added in the continued presence of amiloride to measure the UTP-activated Cl^– secretory response (n = 12). In a separate experiment, perfusion of a low Cl^– solution containing amiloride with 100 μM ISO in a subset of the subjects tested in A–C (n = 6). (E and F) Typical traces showing sequential addition of amiloride, a low Cl^– solution with 100 μM ISO, and UTP on low- and high-level E2 days, respectively, in CF subjects. (G) Paired mean basal CF nasal PDs measured on low– and high–E2 level days (n = 10). (H) Paired mean changes in nasal PD in CF patients following amiloride addition and following perfusion of a low Cl^– solution containing 100 μM ISO and a low Cl^– solution containing 100 μM UTP added in the continued presence of amiloride to measure the UTP-activated Cl^– secretory response (n = 10). *P < 0.05 difference in UTP secretion between low– and high–E2 level days. ^†P < 0.05 compared with non-CF.

Figure 3. E2 inhibits ASL volume homeostasis in CF airways.

(A) XZ confocal images of ASL (red) 48 hours after phasic shear stress (0.6 dynes/cm²) in non-CF (top) and CF cultures (bottom) with 0, 0.1, or 3 nM serosal E2. (B) Mean data taken from A. White bars, non-CF HBECs (n = 6); black bars, CF HBECs (n = 5–7); gray bar, CF HBECs with 5 U/ml mucosal apyrase (n = 5). *P < 0.05 compared with 0 nM E2. ^†P < 0.05 compared with non-CF HBECs. Scale bars: 7 μm.

Figure 2. E2-mediated inhibition of ASL volume homeostasis is due to changes in E2 concentrations, not ER expression levels.

(A and B) Real-time qPCR analysis in non-CF and CF airways, respectively, using primers directed to ERα and ERβ. For ERα, DNAs were obtained from 12 non-CF and 12 CF donors (for each, n = 6 males, white bars, and 6 females, black bars). For ERβ, cDNA was obtained from 6 non-CF and 6 CF donors (for each, n = 3 males, open bars, and 3 females, closed bars). In both cases, expression was normalized to peptidylprolyl isomerase A (PPIA). ERβ was not detectable by either standard PCR (not shown) or qPCR. (C and D) Mean non-CF and CF changes in ASL height, respectively, as measured by confocal microscopy following 100 μM ATP addition ± 10 nM serosal E2 to HBECs from male (white bars) and female donors (black bars). All n = 4. (E) Dose-response curve for the inhibition of ATP-mediated ASL secretion in non-CF HBECs (ΔASL height before and after 10 minutes application of 100 μM ATP) following serosal E2 addition over the range of 0.01 to 1000 nM E2 in non-CF cultures (n = 6). (F) ΔASL height before and after 10 minutes of 100 μM ATP or ADO addition. Hormones were added serosally at 10 nM prior to ATP/ADO addition at 10 μM. All n = 5–7. *P < 0.05 versus 0 nM E2. ^†P < 0.05 versus ATP alone. Progest., progesterone; testost, testosterone.

Figure 4. Transient expression of ERα but not ERβ inhibits ATP-dependent increases in intracellular Ca²⁺ when activated by E2 in BHK cultures.

(A and B) Images of fura-2–loaded BHK cells (green) expressing either ERα or ERβ, respectively, linked to mOr. (C) Mean fura-2 emission ratio over time simultaneously imaged in nontransfected BHK cells (squares) and neighboring cells expressing mOr-ERα (circles) following a 10-minute pretreatment with 10 nM E2, then a 10 μM ATP addition (n = 7). (D) Mean fura-2 emission ratio over time simultaneously imaged in nontransfected BHK cells (squares) and mOr-ERα–expressing cells (circles) (n = 9) following a 30-minute pretreatment with ICI₁₈₂₇₈₀, then 10 minutes with 10 nM E2 followed by a 10 μM ATP addition. (E) Mean ATP-induced changes in fura-2 emission without (white bars) and with (black bars) 10 nM E2 in nontransfected cells (n = 6) and following transfection with mOr (n = 3), mOr-ERα (n = 7), mOr-ERα in the presence of ICI₁₈₂₇₈₀ (n = 7), and mOr-ERβ (n = 5). *P < 0.05 compared with control. Scale bars: 10 μm.

Figure 5. E2 does not induce P2Y₂-R internalization.

BHK cells were transfected with HA-tagged P2Y₂-R (green) ± mOr-ERα (red) and fixed in PFA after E2/ATP addition. (A–C) Sequential images of HA–P2Y₂-R before E2 addition, 30 minutes after 10 nM E2 exposure, and 30 minutes after the addition of 100 μM ATP in the presence of E2, respectively. (D–F) P2Y₂-R cotransfected with mOr-ERα before and after 30-minute 10 nM E2 exposure and 30 minutes after the addition of 100 μM ATP in the presence of E2. (G) Bar graph quantifying HA–P2Y₂-R internalization to the area measured. White bars, HA–P2Y₂-R alone; black bars, HA–P2Y₂-R and ERα. Data are from transfections performed on 3 separate occasions. Scale bars: 10 μm. *P < 0.05 versus E2 alone.

Figure 6. E2 inhibits Ca²⁺ influx in airway epithelia.

Non-CF HBECs and JME CF cells were incubated with vehicle (white bars) or 10 nM E2 (black bars) unless otherwise specified. (A) Mean changes in fura-2 emission ratio in HBECs exposed to ATP (10 μM) or UDP (1 mM) ± E2 (both n = 6). (B) Mean changes in fura-2 emission ratio (340/380 nm) in non-CF HBECs (open symbols; n = 6) and CF JME cells (closed symbols; n = 4–6) exposed to 0.01–100 nM serosal E2 followed by 10 μM mucosal ATP. (C) [³H]IP release from JME cells before and after 10 μM ATP addition ± E2. (D) Mean changes in fura-2 emission ratio following 1 μM thapsigargin addition ± E2 to JME cells in a modified Ringer bath solution with 0 mM Ca²⁺ and 2 mM EGTA (all n = 6). (E) Mean change in fura-2 emission over time following 10 μM ATP addition in JME cells ± E2 with 2 mM external Ca²⁺ (n = 9). (F) Fura-2 emission over time following 10 μM ATP addition in JME cells ± E2 with 0 Ca²⁺ and 2 mM EGTA (n = 9). (G) Mean I_SC in non-CF HBECs ± E2 (10 nM). I_sc was measured following mucosal amiloride (100 μM) treatment, after which Cl^– secretion was elicited either with UTP (100 μM) or ionomycin (1 μM) (all n = 16). *P < 0.05 compared with control.

Figure 7. Antiestrogens reverse the adverse effects of E2 on ASL volume homeostasis.

(A) Mean change in fura-2 emission ratio in JME cells exposed to ATP (10 μM) and vehicle or ATP and either 10 nM E2, E2 and 1 μM ICI₁₈₂₇₈₀, ICI₁₈₂₇₈₀, E2 with 10 μM TXN, and ICI₁₈₂₇₈₀, or 10 μM TXN. All n = 6. (B and C) ASL height measured by XZ confocal microscopy in non-CF and CF HBECs, respectively. 200 μM mucosal ATP addition (squares); 200 μM mucosal ATP and 200 μM mucosal TXN (circles); 200 μM mucosal ATP and 200 μM mucosal TXN and 10 nM serosal E2 (triangles). (D) ASL height over time in non-CF (closed symbols) and CF (open symbols) HBECs with TXN ± bumetanide. Squares, 200 μM mucosal TXN; triangles, 200 μM mucosal TXN and 10 μM serosal bumetanide. All data shown as mean ± SEM. *P < 0.05 compared with ATP or TXN alone. ^†P < 0.05 versus 0 minutes.