G protein-coupled estrogen receptor inhibits the P2Y receptor-mediated Ca2+ signaling pathway in human airway epithelia
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P2Y receptor activation causes the release of inflammatory cytokines in the bronchial epithelium, whereas G protein-coupled estrogen receptor (GPER), a novel estrogen (E2) receptor, may play an anti-inflammatory role in this process. We investigated the cellular mechanisms underlying the inhibitory effect of GPER activation on the P2Y receptor-mediated Ca2+ signaling pathway and cytokine production in airway epithelia. Expression of GPER in primary human bronchial epithelial (HBE) or 16HBE14o- cells was confirmed on both the mRNA and protein levels. Stimulation of HBE or 16HBE14o- cells with E2 or G1, a specific agonist of GPER, attenuated the nucleotide-evoked increases in [Ca2+]i, whereas this effect was reversed by G15, a GPER-specific antagonist. G1 inhibited the secretion of two proinflammatory cytokines, interleukin (IL)-6 and IL-8, in cells stimulated by adenosine 5′-(γ-thio)triphosphate (ATPγS). G1 stimulated a real-time increase in cAMP levels in 16HBE14o- cells, which could be inhibited by adenylyl cyclase inhibitors. The inhibitory effects of E2 or G1 on P2Y receptor-induced increases in Ca2+ were reversed by treating the cells with a protein kinase A (PKA) inhibitor. These results demonstrated that the inhibitory effects of G1 or E2 on P2Y receptor-mediated Ca2+ mobilization and cytokine secretion were due to GPER-mediated activation of a cAMP-dependent PKA pathway. This study has reported, for the first time, the expression and function of GPER as an anti-inflammatory component in human bronchial epithelia, which may mediate through its opposing effects on the pro‐inflammatory pathway activated by the P2Y receptors in inflamed airway epithelia.
KeywordsGPER P2Y receptor signaling pathway Human bronchial epithelial cells Calcium signaling cAMP
Estrogen (E2) is an important hormone that protects the lungs from inflammatory damage. Clinical observations suggested that reduced E2 levels were associated with greater risks of lung pathologies in menopausal women [19, 36]. However, the detailed anti-inflammatory role played by E2 and its pathophysiological mechanism are still unknown. In addition to the classical nuclear hormone receptors, ERα and ERβ, a novel E2 receptor and a G protein-coupled estrogen receptor (GPER), were recently identified [35, 43]. Despite the accumulating body of evidence indicating that the rapid, nongenomic actions of E2 observed in the epithelia are mediated via GPER, few studies have investigated the specific role of GPER in inflammatory airway diseases [31, 32].
Extracellular nucleotide release and the subsequent activation of P2Y receptors have been implicated in the pathogenesis of several inflammatory lung disorders, such as asthma . During airway inflammation, damage to the surface epithelium is due to the secretion of eosinophil-derived, highly toxic, cationic proteins, such as major basic protein (MBP). Our recent study demonstrated that when human bronchial surface epithelia are chemically damaged by poly-l-arginine as a surrogate of MBP , nucleotides, such as ATP and UDP, are released into the extracellular medium. The extracellular nucleotides then activate cell surface P2Y receptors to release two proinflammatory cytokines, interleukin (IL)-6 and IL-8, via a Ca2+-dependent process .
To the best of our knowledge, no reports have determined whether GPER is expressed in airway epithelia or whether GPER plays a role in the regulation of P2Y receptor-mediated Ca2+ signaling and cytokine secretion in airway epithelia. Therefore, we examined the expression of GPER and its subcellular localization in human bronchial epithelia. We also characterized the cross talk between the GPER and P2Y receptor signaling pathways and its implications on the anti-inflammatory role of GPER.
Materials and methods
Solutions and chemicals
Krebs-Henseleit (KH) solution and the nominally Ca2+-free solution were prepared as previously described . Membrane permeant acetoxymethyl (AM) ester forms of Fura-2 and Pluronic F127 were obtained from Invitrogen (Carlsbad, USA). Uridine 5′-triphosphate (UTP), uridine 5′-diphosphate (UDP), adenosine 5′-(γ-thio)triphosphate (ATPγS), forskolin, poly-l-arginine hydrochloride, SQ 22536, U73122, E2, and G1 were obtained from Sigma-Aldrich (St. Louis, USA). G15 was obtained from Tocris (Bristol, UK). H89 dihydrochloride and MDL 12330A were obtained from Calbiochem (La Jolla, USA). All other general laboratory reagents were obtained from Sigma-Aldrich, and all cell culture reagents were obtained from Invitrogen.
The 16HBE14o- cell line was maintained in minimum essential medium (MEM) supplemented with 10 % fetal bovine serum, 1 % penicillin/streptomycin, and 1 % glutamine (Invitrogen, Carlsbad, CA) and cultured as described previously . In some experiments, MEM with no phenol red (Invitrogen) was applied. Primary HBE cells were obtained from ScienCell Research Laboratories (Carlsbad, USA) and cultured using Bronchial Epithelial Cell Medium (ScienCell Research Laboratories) following the commercial protocol described previously .
RNA extraction, reverse transcription PCR, and real-time PCR
Total RNA was extracted with TRIzol Reagent (Invitrogen) and reverse transcribed to cDNA using iScript™ Reverse Transcription Supermix (Bio-Rad Laboratories, Hercules, USA). Reverse transcription PCR (RT-PCR) was performed with TaKaRa Taq™ DNA polymerase. Real-time PCR was performed with an Applied Biosystems Power SYBR Green PCR Master Mix (Invitrogen) on a ViiA™ 7 real-time PCR system. GPER primer sequences were as follows: forward primer, 5′-TCTACACCATCTTCCTCTTCC-3′; and reverse primer, 5′-GTAGCGGTCGAAGCTCATCC-3′. The RT-PCR products were characterized using 2 % agarose gel electrophoresis. Relative expression of GPER was normalized to GAPDH and determined with the Pfaffl method . Each run of PCR included a nontemplate control and a sample without reverse transcriptase.
Western blotting was performed as described previously . Cells grown in culture dishes were lysed on ice in Cytobuster™ Protein Extraction Reagent (Merck Millipore, Billerica, USA), supplemented with a protease inhibitor cocktail (no. 78429, Thermofisher Scientific, Waltham, USA) and a phosphatase inhibitor cocktail (Merck Millipore). Protein samples (20 μg per lane) were transferred to polyvinylidene fluoride (PVDF) membranes (Immobilon-P, Merck Millipore) and immunoblotted with GPER rabbit polyclonal antibody (N-15)-R (1:500; Santa Cruz Biotechnology, Santa Cruz, USA). Blocking peptide (sc-38525 P, Santa Cruz Biotechnology) was used for GPER antibody preabsorption, and mouse monoclonal antibody to GAPDH was used as a loading control. All blots were developed using an enhanced chemiluminescence detection system (Merck Millipore). The apparent molecular masses were calculated using prestained sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) midrange protein markers (no. HM0671, Hou-Bio Life Technologies, Hong Kong).
16HBE14o- or primary HBE cells grown on coverslips in four-well plates were rinsed with phosphate-buffered saline (PBS) and fixed in 4 % paraformaldehyde for 10 min at room temperature. Cells were blocked using PBS with 10 % normal horse serum and 0.1 % Triton X-100 for 1 h and incubated with GPER (N-15)-R rabbit polyclonal antibody (1:60; sc-48525-R, Santa Cruz Biotechnology) overnight at 4 °C . After washing, Alexa Fluor® 488 donkey anti-rabbit IgG (H+L) was added (1:300, Thermofisher Scientific, Waltham, USA). The coverslips were mounted using mounting medium with 1.5 μg/ml 4′,6-diamidino-2-phenylindole (DAPI). Images were captured using a FluoView™-FV1000 confocal microscope (Olympus, Center Valley, USA). In some experiments, cells were co-incubated with purified mouse anti-E-cadherin (1:200; no. 610181, BD Biosciences, Heidelberg, Germany), purified mouse anti-GM130 (1:200; no. 610823, BD Biosciences), or KDEL antibody (1:500; NBP1-97469, Novus Biologicals, Littleton, USA). Alexa Fluor® 555 donkey anti-rabbit IgG was used for visualization (1:400, Thermofisher Scientific). For the negative control group, GPER antibodies were preabsorbed with specific blocking peptides (sc-48525 P, Santa Cruz Biotechnology).
Small interfering RNA lentivirus packaging and transduction
Lentiviral transfer vectors containing small interfering RNA (siRNA)-targeting GPER were purchased from Applied Biological Materials Inc. (Canada). A lentiviral vector with a scramble siRNA sequence was used as the negative control. The VSV-G-pseudotyped lentiviruses were produced by co-transfecting 293T cells with the transfer vectors and three packaging vectors, pMDLg/pRRE, pRSV-REV, and pCMV-VSVG, by calcium phosphate transfection. At 72 h post-transfection, the cell culture supernatant was collected and filtered through a 0.4-μm filter. The lentivirus was concentrated with centrifugation at 20,000 rpm and resuspended in 1× Tris-buffered saline. For lentiviral transduction, 5 × 103 cells were seeded in 24-well plates, and lentivirus was added to the cells in the presence of 8 μg/ml hexadimethrine bromide (Sigma-Aldrich, St. Louis, USA) overnight. After puromycin selection, the knockdown efficiency of GPER expression was determined by real-time PCR and Western blot analysis.
Measurement of intracellular calcium concentrations
Calcium signals in cells grown on glass coverslips were measured as previously described [47, 48]. Fura-2 ratios were used to represent changes in [Ca2+]i using Felix software (Photon Technology International, Edison, USA ). In Ca2+ imaging experiments, the perfusion chamber was mounted on an inverted microscope (Olympus IX70, USA) equipped with a scientific CMOS camera (pco.edge 5.5; PCO AG, Kelheim, Germany). Images were digitized and analyzed using MetaFluor Imaging Software (v7.5, Molecular Devices, USA). The data were also shown quantitatively as a change in Fura-2 ratios.
The manganese quench technique was used to estimate calcium influx [15, 45]. 16HBE14o- cells were loaded with Fura-2 as previously described. Since Mn2+ has a similar permeability as Ca2+ through most plasma membrane Ca2+ channels and quenches Fura-2 fluorescence at all excitation wavelengths, Ca2+ influx can be estimated by the Mn2+ quench of Fura-2 fluorescence at the Ca2+-insensitive 360-nm excitation wavelength. During the measurement, cells were treated with 10-μM UTP in the absence (nucleotide alone) or presence of E2 (100 nM) or G1 (10 nM) for 10 min. Then 1-mM MnCl2 was added in perfusion solution to observe the extent of Mn2+ entry. The rate of Mn2+ quenching was assessed by measuring the change of slope of Fura-2 fluorescence decrease before and after the addition of Mn2+ application (using Originlab 8 software, Northampton, USA), as well as the percentage decrease of Fura-2 fluorescence 120 s after Mn2+ application [6, 29, 41].
Monitoring STIM1 oligomerization via FRET microscopy
FRET microscopy was used to monitor the dynamic oligomerization of stromal interaction molecule 1 (STIM1) in 16HBE14o- cells. Plasmids expressing N-terminally tagged cyan fluorescent protein (CFP)- and yellow fluorescent protein (YFP)-STIM1, pEX-SP-CFP-STIM1, and pEX-SP-YFP-STIM1 were obtained from Addgene (Cambridge, USA). Cells were incubated in Ca2+-free Hanks’ Balanced Salt Solution (HBSS) with 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) during imaging experiments. E2, G1, or dimethyl sulfoxide (DMSO) vehicle in HEPES-HBSS modified was perfused into the imaging chamber using a perfusion pencil (AutoMate Scientific, Berkeley, USA) from 0 to 15 min, followed by the addition of ATP to the perfusion solution to deplete Ca2+ in the ER. Images were captured using a Nikon Eclipse Ti microscope with a SPOTS RT3 camera (SPOT Imaging Solutions, Sterling Heights, USA). An ND8 filter was used during the experiment to reduce photobleaching. Each set of images (CFP, YFP, and FRET channels) was taken at 0.2 Hz using MetaFluor 7.8 software. The captured images were analyzed with ImageJ software (NIH, Bethesda, USA) using the pixel-to-pixel comparison method . Sensitized emission was employed for the bleed-through correction. These specific bleed-throughs were obtained on a pixel-to-pixel basis from cells transfected with CFP-STIM1 or YFP-STIM1 alone. In our experimental settings, specific bleed-throughs for CFP and YFP were 0.696 and 0.064, respectively. To reduce photobleaching errors, N FRET measurements were adopted as described .
Quantitative measurement of STIM1 puncta formation
The formation STIM1 puncta after ER Ca2+ depletion by ATP stimulation was measured as described . In brief, 16HBE14o- cells were transfected with YFP-STIM1 (Addgene, Cambridge, USA). Transfected cells were stimulated with ATP (10 μM) in the absence of extracellular Ca2+ in HEPES-HBSS solution. Single and isolated cells were selected for analyses. Z-stacks of images were acquired in a 0.225-μm separation. A Z-slice image near the close proximity to the attachment surface was selected for puncta analysis (~225 nm thickness) by ImageJ (NIH). YFP puncta were determined by particle analysis plugin, and fluorescent intensity greater than the background by three standard deviations was measured. Those with size less than 0.2 μm2 and greater than 2.5 μm2 were excluded for the analyses. In some experiments, transfected cells were pretreated with G1 (10 nM) or E2 (100 nM) before stimulation with ATP.
Real-time measurement of cAMP levels
CFP-Epac-YFP, an Epac-based polypeptide FRET reporter , was used to monitor real-time cyclic adenosine monophosphate (cAMP) changes in 16HBE14o- cells. The experiments were performed using the MetaFluor Imaging system (with the FRET module). Cells were transfected with the Epac-based cAMP sensor and excited at 436-nm wavelengths. CFP and YFP images were simultaneously recorded by the imaging setup equipped with the photometrics DV2 emission splitting system (Photometrics, Tucson, USA) including two emission filters (470/30 nm for CFP; 535/30 nm for FRET). Acquired fluorescence images were background subtracted, and real-time cAMP levels were represented by normalizing the CFP/FRET emission ratios as described previously [20, 24]. Images were digitized and analyzed using MetaFluor imaging software.
16HBE14o- cells were plated 24 h before the experiment into 24-well culture plates at a concentration of 5 × 104 cells/well. Agonist-induced inositol-1-phosphate (IP1) accumulation in 16HBE14o- cells was quantified using the Cisbio IP-One kit (Cisbio Bioassays, Codolet, France) according to the manufacturer’s instructions .
Quantification of IL-6 and IL-8 secretion
Quantification of IL-6 and IL-8 secretion was performed using an enzyme-linked immunosorbent assay (ELISA) . Cells were grown in 24-well culture plates. Cell-free supernatants were collected from control and treated cells and analyzed using a commercially available ELISA kit specific for IL-6 (eBioscience, San Diego, USA) and IL-8 (BD Biosciences, San Diego, USA) according to the manufacturers’ protocols. All experiments were performed in duplicate.
Data were expressed as the mean ± the standard error of the mean (SEM), and values of n referred to the number of independent experiments for each group. Statistical comparisons between original data were performed using the Student’s t test and analysis of variance (ANOVA) where appropriate. P < 0.05 was considered to be statistically significant.
Expression and subcellular localization of GPER in human bronchial epithelial cells
To further characterize the subcellular localization of GPER, double immunofluorescence labeling was used to determine possible colocalization between GPER and subcellular fractions, such as the endoplasmic reticulum (ER), Golgi apparatus, and plasma membrane, in 16HBE14o- cells (Fig. 2c). The results showed that GPER did not colocalize with Golgi (anti-GM130), while there was a very small amount of overlap between the GPER and the ER (anti-KDEL). In contrast, partial colocalization was observed between GPER and the plasma membrane (anti-E-cadherin) in 16HBE14o- cells.
Inhibitory effects of E2 or the GPER agonist, G1, on nucleotide-induced Ca2+ signaling in HBE cells
Various concentrations of E2 and G1 were used to examine their inhibitory effects on P2Y receptor-mediated Ca2+ signaling in 16HBE14o- cells. The 16HBE14o- cells were pretreated with E2 (1–300 nM) or G1 (0.01–100 nM) for 10 min and then stimulated by 10-μM UTP in the presence of E2 or G1. Both E2 and G1 inhibited the UTP-induced increases in Ca2+ in a concentration-dependent manner (Fig. 3i, j). The half maximal inhibitory concentration (IC50) values of E2 and G1 were 12.42 and 0.58 nM, respectively.
In addition to using a specific antagonist, we also used lentiviral vectors expressing siRNA to downregulate the GPER gene in 16HBE14o- cells. The knockdown efficiency of GPER was examined on both the mRNA and protein levels (Fig. 4d, e). The expression level of GPER in 16HBE14o- cells transfected with siRNA targeting GPER (siGPER) was 43.9 % lower than those transfected with a negative control siRNA encoding a scrambled sequence (SCR; Fig. 4f). The presence of GFP did not disturb Fura-2 fluorescence during the measurement of [Ca2+]i . The successfully transfected cells were selected for calcium measurements. The data show that 10-nM G1 inhibited UTP-induced increases in Ca2+ in the SCR control group (Fig. 4g), whereas no significant inhibitory effect was observed in the siGPER group treated with G1 (Fig. 4h, i). These results indicate that the inhibitory effect of G1 was mediated by GPER.
Effects of E2 and G1 on nucleotide-induced Ca2+ release and influx
The manganese quench technique was also applied to report calcium influx through plasma membrane channels. The quenching of Fura-2 fluorescence by Mn2+ was measured at the Ca2+-independent excitation wavelength of Fura-2 (360 nm). When preincubating the cells with 100-nM E2 for 10 min, the rate of change on Mn2+ quenching was significantly decreased compared to untreated or G1 pretreated 16HBE14o- cells (Fig. 5f, g). Similarly, the percentage decrease of Fura-2 fluorescence 120 s after Mn2+ application also dropped significantly in E2 pretreated cells but not in G1 pretreated cells (Fig. 5h). These results indicate that E2, but not G1, blocked calcium influx through the plasma membrane.
Calcium release from the ER mainly occurs via inositol 1,4,5-trisphosphate receptors (IP3R) in human airway epithelial cells, including 16HBE14o- cells . The activation of various subtypes of P2Y receptors causes an increase in Ca2+ via the phospholipase C (PLC)-IP3 signaling cascade . Because the lifetime of IP3 within the cell before it is transformed into IP2 and IP1 is very short, IP1 accumulation levels can be used to represent IP3 levels in cells. To induce IP1 accumulation by activating the P2Y receptor-mediated signaling pathway in 16HBE14o- cells, 10-μM UTP (Fig. S1a) or 100-μM UDP (Fig. S1b) was used. Dimethyl sulfoxide (DMSO), the solvent used for E2 and G1, did not affect IP1 accumulation. UTP- or UDP-induced IP1 could be blocked by U73122 (10 μM), an inhibitor of PLC, whereas E2 (10 and 100 nM) or G1 (10 nM) had no effect on UTP- or UDP-induced IP1 accumulation.
E2- and G1-induced cAMP production
To demonstrate the involvement of adenylyl cyclase (AC) in cAMP production, two AC inhibitors, MDL 12330A and SQ 22536, were used. G1-induced cAMP production was significantly inhibited by 10-min treatments with MDL 12330A (0.1, 1, and 10 μM) in a concentration-dependent manner (Fig. 7c). Similar results were obtained with SQ 22536 (Fig. 7d). These results suggest that GPER is coupled to the activation of AC, likely via the Gs alpha subunit, to stimulate an increase in cAMP levels in 16HBE14o- cells.
The role of PKA in E2- and G1-mediated inhibition of calcium increases
We next determined whether the cAMP-dependent pathway was involved in the inhibitory effects of E2 and G1 on the P2Y-induced increase in Ca2+ in 16HBE14o- cells. H89, a PKA inhibitor, was used to inhibit downstream signaling targets of cAMP. The inhibitory effects of G1 (10 nM) on 10-μM UTP- or 100-μM UDP-induced increases in Ca2+ were reversed by co-incubation of the cells with H89 (10 μM) for 10 min (Fig. 7e). Similar results were obtained with 100-nM E2 (Fig. 7f). These results demonstrate that the inhibitory effects of E2 and G1 on P2Y receptor-induced Ca2+ signaling are mediated via the activation of a cAMP-dependent PKA pathway.
Effects of the GPER agonist, G1, on nucleotide- or poly-l-arginine-induced cytokine production in HBE cells
GPER is known to play important roles in multiple tissues, including the heart, brain, lung, liver, skeletal muscle, and kidney . However, few reports have described the expression and function of GPER in airway epithelial cells. Only one study described the relatively high expression of GPER in human nonsmall cell, lung cancer cell lines compared to immortalized normal lung bronchial epithelial cells . This study therefore reports for the first time the expression and function of GPER as an anti-inflammatory component in human bronchial epithelia and highlights that GPER likely serves this role through its opposing effects on the proinflammatory pathway activated by the P2Y receptors in inflamed airway epithelia.
The mRNA and protein expression levels of GPER in primary HBE and 16HBE14o- cells were confirmed by RT-PCR and Western blotting, respectively. Although GPER localizes to the ER  and plasma membrane , its subcellular localization is still controversial. Overall, the localization of GPER appears to vary depending on the cell type. Our immunofluorescence studies showed partial colocalization between the GPER and the plasma membrane with no overlap between GPER and Golgi, or nucleus. Besides, a very small amount of GPER was localized in ER. These results were similar to those observed in osteocyte-like MLO-Y4 cells and transfected HEK-293 cells [17, 34]. Different tissues or cell types may have different subcellular GPER localizations, which may be due to the different roles GPER plays in various cell types, such as cell proliferation, apoptosis, and immune responses . However, it should be noted that, even in the same cells, GPER could change its location via endocytotic processes. Two reports have demonstrated that GPER can be trafficked intracellularly from the plasma membrane [8, 38]. This unique mechanism could decrease the amount of GPER at the plasma membrane and protect cells from chronic signaling. Thus, we could not exclude this possibility, but the exact details of this intriguing membrane receptor trafficking pathway in human bronchial epithelia require further study.
In this study, activation of GPER by G1 did not alter basal [Ca2+]i levels, but it did significantly inhibit P2Y receptor-mediated increases in Ca2+. This inhibitory effect was not P2Y receptor subtype-specific, because G1 inhibited the Ca2+ responses elicited by different P2Y receptor subtype ligands (e.g., UTP, UDP, and ATPγS). The inhibitory effect could be reversed when pretreating with GPER-specific antagonist, G15. Besides, siRNA knockdown of GPER was also applied to further confirm the role of GPER. Various transfect reagents (e.g., lipofectamine 2000, lipofectamine RNAiMax, siPORT NeoFX, DharmaFECT 1 and 4) have been applied in our experiment for GPER silencing; however, the knockdown efficiency was low and inconsistent (data not shown). Therefore, lentiviral-mediated silencing approach was adopted and a stable silenced cell line was generated with better knockdown efficiency on GPER. To reveal whether G1 inhibited intracellular increases in Ca2+ by blocking Ca2+ release and/or Ca2+ influx, we characterized the two phases of Ca2+ increase by perfusing cells with a Ca2+-free Krebs-Henseleit Buffer, followed by a Ca2+-containing solution. The results showed that E2 inhibited both P2Y receptor-mediated Ca2+ release and Ca2+ influx, whereas G1 only inhibited Ca2+ release. However, the observed difference in Ca2+ signals could be due to regulation of Ca2+ pumps. The inhibitory effect of E2 on Ca2+ influx was further confirmed by the data obtained from Mn2+ quench experiments. Taken together, these data suggest that the activation of GPER only inhibits P2Y-activated IP3-mediated Ca2+ release, whereas classical E2 receptors activated by E2 played a role in regulating Ca2+ influx. FRET microscopy further confirmed that the inhibitory effect of E2, but not G1, on SOC influx was due to the inhibition of STIM1 oligomerization. Our findings were similar to those reported recently , which showed that E2 can signal nongenomically by inhibiting basal phosphorylation of STIM1, leading to a reduction of SOC entry in human airway cells.
The inhibition of Ca2+ release by G1 could occur via different pathways. For example, GPER activation might block the activity of PLCβ to diminish the synthesis of IP3, inhibit the activity of IP3R to release Ca2+ from ER, or stimulate Ca2+ uptake into stores by activating the endoplasmic Ca2+-ATPase pump. To investigate the detailed mechanism underlying the inhibitory effects of E2 and G1 on P2Y receptor-mediated Ca2+ mobilization, we conducted another series of studies to examine whether E2 or G1 could inhibit IP3 production. We measured IP1 accumulation to determine IP3 levels. IP1 accumulation induced by both UDP and UTP was significantly inhibited by the PLC inhibitor, U73122, whereas the addition of E2 or G1 showed no inhibitory effects. This result indicates that the E2- or G1-mediated inhibition of P2Y receptor-mediated Ca2+ signaling was not related to a change in IP3 levels. Thus, the observed inhibition may be due to the inhibition of IP3 independent of Ca2+ release. Alternatively, it may be due to an unidentified signaling pathway or molecule that interferes with the interaction between IP3 and IP3R .
GPER couples to different signaling pathway(s), including the cAMP/PKA pathway . Our data suggest that GPER in human bronchial epithelia was coupled to AC, resulting in an increase in cAMP levels. Notably, blocking the downstream target of cAMP with H89 reversed the inhibitory effect of G1 on P2Y receptor-mediated Ca2+ signaling, and cAMP-dependent protein kinase reportedly inhibits IP3-induced Ca2+ release in human bone marrow cells . The cAMP/PKA pathway may inhibit receptor-operated calcium entry (ROCE) via transient receptor potential canonical channel 6 (TRPC6). TRPC6 is expressed in both undifferentiated and differentiated primary HBE cells . Calcium influx mediated by TRPC6 is functionally coupled to calcium-activated chloride channel activity in human airway epithelial cells  and can be regulated by P2Y receptor activation in mouse podocytes . A recent study suggests that the cAMP/PKA signaling pathway can inhibit endothelin type A receptor-mediated ROCE via TRPC6 by phosphorylation of Ser28 site in human embryonic kidney 293 cells . Although G1 did not have any significant effect on nucleotide-mediated Ca2+ influx, we did not explicitly examine P2Y receptor-mediated ROCE in this study. It would be interesting for future research to examine if GPR30 can inhibit P2Y receptor-mediated ROCE via TRPC6 in human airway epithelia. In addition to PKA, Epac is another downstream target of cAMP that transduces diverse cellular actions [7, 9]. The cAMP increases evoked by G1 are sufficient to activate Epac. Interestingly, our previous study demonstrates that both Epac 1 and Epac 2 are expressed in 16HBE14o- cells . Therefore, we could not exclude the possibility that some of the observed inhibitory effects were mediated through activation of Epac. Moreover, our recent study demonstrates that the proinflammatory effect of nucleotides is mediated via an increase in [Ca2+]i after P2Y receptor activation. Treating 16HBE14o- cells with the intracellular Ca2+ chelator, BAPTA-AM, but not H89, inhibited P2Y receptor-mediated IL-6 and IL-8 secretion . Taken together, GPER likely inhibits the P2Y receptor-mediated inflammatory response by downregulating [Ca2+]i in human airway epithelia. A recent study reported that Ca2+-dependent calmodulin can regulate GPER-dependent signaling at the receptor level . Therefore, a P2Y receptor-mediated increase in Ca2+ could, in turn, regulate GPER function, but the details of the possible cross talk between the two receptors require further investigation. 16HBE14o- cells were cultured in MEM without phenol red in some experiments since phenol red may serve as a weak estrogen mimic. However, no significant differences have been observed in terms of GPER expression and the inhibitory effect of G1 on P2Y receptor-mediated cytokine secretion and Ca2+ increase (data not shown) in cells cultured in MEM with or without phenol red.
In summary, this study characterizes the expression, localization, and role of GPER, as well as its interaction with P2Y receptors, that were co-expressed in human bronchial epithelia. Activation of GPER by E2 or its specific agonist, G1, rapidly attenuated a nucleotide-evoked increase in Ca2+, whereas the specific GPER antagonist, G15, reversed this GPER-mediated inhibition. Furthermore, E2 and G1 also inhibited nucleotide-induced cytokine release. The inhibitory effects on P2Y receptor-mediated Ca2+ mobilization and cytokine secretion are likely due to GPER-mediated activation of a cAMP-dependent PKA pathway in human bronchial epithelia (Fig. 8d).
We thank Dr. D. C. Gruenert (University of Vermont, Burlington, USA) for the generous gift of the 16HBE14o- cells and Dr. K. Jarlink (The Netherlands Cancer Institute, Amsterdam, the Netherlands) for supplying the Epac sensor. We thank Dr. Bernard Lam, Department of Physiology, National University of Singapore, and Prof. Brian B. J. Harvey, Department of Molecular Medicine, Royal College of Surgeons in Ireland, Ireland, for their expert advice.
This work was supported by a grant from the Research Grants Council General Research Fund (Ref. No. 466611) awarded to W.H. Ko.
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