The dopamine D₁ receptor is expressed and induces CREB phosphorylation and MUC5AC expression in human airway epithelium

Protease inhibitor cocktail III was purchased from EMD Millipore (Billerica, MA). Antibiotic-antimycotic mix, DMEM/F-12 medium, fetal bovine serum (FBS), and RPMI-1640 medium were purchased from Thermo Fisher Scientific (Waltham, MA). A68930 and SCH39166 were purchased from Tocris Bioscience (Bristol, UK). All other chemicals were obtained from Sigma-Aldrich (St. Louis, MO) unless otherwise stated.

Primary cultured normal human bronchial epithelial cells (CC-2541; Lonza, Walkersville, MD) were grown in Clonetics™ BEGM BulletKit (CC-3170, Lonza) supplemented with the following growth supplements: bovine pituitary extract, hydrocortisone, human epidermal growth factor, epinephrine, transferrin, insulin, retinoic acid, triiodothyronine, and gentamicin/amphotericin-B at the concentrations recommended by the manufacturer. 16HBE14o- cells, a human bronchial epithelial cell line which was kindly gifted from Dr. Tilla S. Worgall (Columbia University, New York NY), were grown in minimal essential medium supplemented with 10% FBS and 200 μg/ml geneticin (G418). NCI-H292 cells (CRL-1848; American Type Culture Collection, Manassas, VA), a human pulmonary muco-epidermoid carcinoma cell line, were cultured in RPMI-1640 medium containing 5% FBS. Primary cultured human airway smooth muscle cells (HASM; cc-2576, Lonza) were grown in DMEM/F12 culture medium, supplemented with 10% FBS and an antibiotic-antimycotic mix (100 units/ml penicillin G sodium, 100 μg/ml streptomycin sulfate, 0.25 μg/ml amphotericin B). All the cells were incubated at 37°C in humidified 95% air/5% CO₂.

Studies were approved by Columbia University’s Institutional Review Board (IRB) and deemed not human subjects research under 45 CFR 46. Human trachea was obtained from discarded regions of healthy donor lungs harvested for lung transplantation at Columbia University. Human tissue was transported to the laboratory in cold (4 °C) M199 cell culture media. The exterior of human trachea was carefully dissected free of adherent connective tissue under a microscope. The tissue sample was used for immunohistochemistry and immunoblot.

Human tracheal rings were fixed with 4% paraformaldehyde/1% glutaraldehyde in 0.1 M phosphate buffer for 4 h at 4°C and were then dehydrated through a graded ethanol series. The tracheal rings were embedded in paraffin and cut into 10-μm-thick sections. The tracheal ring sections were deparaffinized in xylene and rehydrated in descending grades of alcohol. Heat-mediated antigen retrieval was performed in Tris-EDTA buffer (10 mM Tris-base and 1 mM EDTA, pH 9.0) for 2 min using a pressure cooker. Endogenous peroxidase activity was blocked with 3% H₂O₂ for 15 min. Sections were blocked with 10% normal goat serum in phosphate-buffered saline with 0.1% Triton X-100 (PBST) for 30 min, then avidin-biotin blocking was performed as previously described [26]. The slides were kept in humidified chamber with primary antibody against the dopamine D₁ receptor protein (rabbit monoclonal 1:2000; 2192–1, Epitomics, Burlingame, CA) or dopamine D₅ receptor protein (rabbit polyclonal 1:2000; sc-25,650, Santa Cruz Biotechnology, Santa Cruz, CA) in 2% normal goat serum in PBST for overnight at 4 °C. The immunoreactivity of these antibodies directed against the dopamine D₁ or D₅ proteins were previously confirmed in control tissues during immunohistochemistry [20]. A parallel tracheal ring section was incubated with an isotype-specific rabbit IgG antibody (Thermo Fisher Scientific) as a negative control. The slides were then washed three times with PBST and primary antibodies were detected using biotinylated anti-rabbit antibodies (Vector Laboratories, Burlingame, CA) at a concentration of 1:200. After incubation with ABC-HRP complex (Vector Laboratories) for 30 min, the antigen-antibody complex was visualized with the peroxidase substrate kit (DAB) (SK-4100, Vector Laboratories). The sections were counterstained with hematoxylin (Vector Laboratories), dried, dehydrated in ascending grades of alcohol, and cover slipped using Poly-mount mounting medium (Polysciences, Warrington, PA).

Freshly dissected native human airway epithelium was homogenized (Tekmar Ultra Turrax T25 high-speed homogenizer set at top speed for 30 s) in cold (4°C) buffer (50 mM Tris, 10 mM HEPES, pH 7.4, 1 mM EDTA with a 1:200 dilution of protease inhibitor cocktail III). The homogenate was filtered through 125-μm Nitex mesh and centrifuged twice at 500 g for 15 min. The supernatant was transferred into new tubes and centrifuged at 50,000 g for 30 min at 4°C. The final membrane pellet was resuspended in the same buffer for protein concentration determinations and stored at − 80°C.

For analysis of dopamine D₁ receptor expression, confluent cultures of either primary cultured normal human bronchial epithelial cells, 16HBE14o- cells, NCI-H292 cells, or primary cultured human airway smooth muscle cells were rinsed with ice-cold phosphate-buffered saline (PBS), and mechanically scraped from the surface of the T75 culture flask in the presence of protease inhibitor cocktail III. Cells were pelleted (500 g, 10 min, 4 °C) and lysed in ice-cold lysis buffer [20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM Na₂EDTA, 1 mM EGTA, 1% Nonidet P-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na₃VO₄, 1 μg/ml leupeptin, 1 mM phenylmethanesulfonyl fluoride, 1:200 dilution of protease inhibitor cocktail III]. Lysed cells were centrifuged (15,000 g, 15 min, 4 °C) and an aliquot of the supernatant was subjected to protein analysis and storing at − 80 °C. For analysis of CREB phosphorylation, 16HBE14o- cells or NCI-H292 cells were serum-starved for 24 h, and then treated with dopamine D₁ receptor agonist A68930 (1 μM) for indicated times (5–60 min). In separate experiments, NCI-H292 cells were initially pretreated with 10 μM H89 (PKA inhibitor; 30 min) or 5 μM U0126 (MEK inhibitor; 120 min) before treatment of the cells with A68930 (1 μM; 20 min). After treatment, the cells were washed twice with ice-cold PBS, and lysed in ice-cold RIPA buffer (Cell signaling Technology (CST), Danvers, MA) supplemented with 1 mM phenylmethanesulfonyl fluoride and a 1:200 dilution of protease inhibitor cocktail III. Each lysed cell sample was harvested and centrifuged at 15000 g for 15 min at 4 °C, and an aliquot of the supernatant was subjected to protein analysis. The protein concentration of each sample was determined using Pierce BCA reagents (Thermo Fisher Scientific), using BSA as a control, and samples were stored at − 80 °C. Each sample was solubilized by heating at 95 °C for 10 min in sample buffer (final concentrations: 50 mM Tris HCl pH 6.8, 2.5% SDS, 6% glycerol, 2.5% 2-mercaptoethanol, and bromophenol blue) before use. Lysates were electrophoresed (10% Mini-Protean TGX™ precast gel; Bio-Rad, Hercules, CA) and transferred to PVDF membranes using a Trans-Blot Turbo™ transfer system (Bio-Rad). The PVDF membrane was blocked for 1 h at room temperature with 5% membrane blocking agent (RPN418; GE Healthcare, Waukesha, WI) in Tris-buffered saline with 0.1% Tween 20 (TBST). Membranes were then probed with antibodies directed against the dopamine D₁ receptor protein (rabbit monoclonal 1:1000; 2192–1, Epitomics) or the dopamine D₅ receptor protein (rabbit polyclonal 1:500; sc-25,650, Santa Cruz Biotechnology) overnight at 4 °C. For the CREB phosphorylation study, the membranes were probed with antibodies directed against the anti-phospho CREB (rabbit monoclonal 1:1000; CST #9198), or anti-CREB (rabbit monoclonal 1:1000; CST #9197) overnight at 4 °C. After washing three times with TBST, membranes were incubated for 1 h at room temperature with HRP-labeled secondary anti-rabbit antibodies (1:5000; GE Healthcare, NA934V). The signals from the immunoreactive bands were detected by ECL Prime (GE Healthcare) and the signal was captured using a chemiluminescent image analyzer (LAS 4000 Mini; GE Healthcare). The same PVDF membranes were stripped and reprobed with the antibody against the GAPDH protein (rabbit monoclonal 1:2000, CST #5174) to demonstrate the variation in protein loading on the gels. For analysis of CREB phosphorylation, the band intensities were measured using Image J software (NIH) and were expressed as a ratio of the phosphorylated/total CREB protein.

Cyclic AMP (cAMP) production in 16HBE14o- and NCI-H292 cell lines was measured using a HitHunter™ cAMP Assay for Small Molecules kit (DiscoverX, Fremont, CA) according to the manufacturer’s instructions. Briefly, the cells grown in white-walled 96-well plates and were washed twice with warm PBS (37 °C). The cells were incubated with dopamine (1 μM), or the dopamine D₁-like receptor agonists (A68930 or SKF38393) (1 μM) for 20 min at 37 °C. The concentration and duration of the dopamine D₁-like receptor agonists used in this study was determined based on previous studies [20, 27]. In separate experiments, the cells were pretreated with the dopamine D₁-like receptor antagonists [SCH23390 (1 μM) or SCH39166 (1 μM)] or vehicle (PBS) for 30 min followed by incubation with A68930 (1 μM) for 20 min at 37 °C. Then the cAMP antibody reagent followed by the cAMP working solution (mixture of enzyme donor/lysis buffer/Emerald II/Galacton) was added to each well and incubated for 60 min at room temperature. Cells were further incubated with the enzyme acceptor reagent for 3 h at room temperature, and luminescence signals were detected using a multimode microplate reader (Appliskan, Thermo Fisher Scientific). The data from the triplicate wells were averaged for each sample.

Cigarette smoke extract (CSE) was prepared by a modification of a previously published method [28]. Smoke of four commercial filtered cigarettes (Marbolo Red, Philip Morris, Richmond, VA; 1.0 mg nicotine; 12 mg Tar) was bubbled through 50 mL of PBS using a vacuum pump. This 100% CSE solution was adjusted to pH 7.4 and filtered through a 0.2 μm-pore filter to remove bacteria and large particles.

NCI-H292 cell viability after 48 h treatment with CSE (10% or 20%) was measured using an MTT assay kit (Cayman Chemical, Ann Arbor, MI) according to the manufacturer’s instructions. Briefly, NCI-H292 cells were seeded in a 96-well plate (5000 cells/well) in RPMI-1640 medium containing 0.5% FBS for 24 h. Then, the cells were incubated with or without CSE (10% or 20%) for 48 h in a final volume of 100 μl/well. Ten microliters of MTT reagent were added to each well and the cells were re-incubated for 4 h at 37 °C in a CO₂ incubator. The medium was aspirated, and 100 μl of the crystal dissolving solution were added to each well. The absorbance of each sample was measured at 570 nm using a multimode microplate reader (Appliskan; Thermo Fisher Scientific). The results were expressed as a percentage of surviving cells over control (no treatment) cells.

Total RNA was extracted from cultured NCI-H292 cells after 48 h treatment with dopamine (1 μM), A68930 (1 μM), isoproterenol (1 μM), or CSE (10%) using the RNeasy Mini Kit (QIAGEN, Valencia, CA). Total RNA was transcribed into cDNA using the ReverTra Ace qPCR RT Kit (Toyobo, Osaka, Japan) in accordance with the manufacturer’s instructions. Quantitative real-time PCR on the CFX96 Real-Time PCR Detection System (Bio-Rad) was performed using Thunderbird SYBR qPCR kit (Toyobo) according to manufacturer’s instruction. Primer sequences for MUC5AC and GAPDH were shown in Table 1. The specificity of amplification was confirmed by melting curve analysis. The Ct value determined by the CFX manager Software (Bio-Rad) for all samples was normalized to the housekeeping gene GAPDH, and the relative fold induction against untreated controls was computed by the comparative Ct (ΔΔCt) method.

Immunofluorescence staining of MUC5AC protein in NCI-H292 cells was carried out according to the previously described method [29] with some modifications. Briefly, NCI-H292 cells were seeded on an 8-chamber microscope slide and serum-starved for 24 h. After starvation, cells were exposed to A68930 (1 μM), dopamine (1 μM), or CSE (10%) for 48 h. Cells were fixed with 4% paraformaldehyde for 15 min at room temperature and washed 3 times with PBS. After permeabilization (0.2% Triton X-100 in PBS for 5 min) and blocking (1% bovine serum albumin in 0.1% Triton X-100 in PBS for 15 min), cells were incubated with Alexa Fluor 488-conjugated MUC5AC antibody (sc-21,701 AF488, Santa Cruz Biotechnology) overnight at 4C. After cells were washed twice with PBS, the slide was coverslipped with ProLong gold antifade-reagent with DAPI (Thermo Fisher Scientific), and visualized with an inverted fluorescent microscope (DMI-4000; Leica Microsystems, Wetzlar, Germany). Digitized images were captured with MetaMorph software (Molecular Devices, Sunnyvale, CA). When capturing the images, we kept constant the duration of image capture (300 ms), the image intensity gain, the image enhancement, and the image black level among the samples.

Initially, we examined the protein expression of the dopamine D₁ and D₅ receptor in human tracheal epithelium by immunohistochemistry. Light microscopic immunohistochemical staining for the dopamine D₁ receptor was observed throughout the epithelial layer of human trachea (indicated by brown color) (Fig. 1a). In contrast, no staining of the dopamine D₅ receptor was detected in the airway epithelial layer (Fig. 1c). Consecutive sections exposed to rabbit IgG isotype-specific control antibodies yielded no staining (Fig. 1b and d).

We further examined whether the dopamine D₁ receptor protein was expressed in human airway epithelial tissue and cells by immunoblot. A single immunoreactive band of the appropriate molecular mass for the dopamine D₁ receptor (75 kDa) was identified in freshly dissected human tracheal epithelium, human pulmonary muco-epidermoid carcinoma cells (NCI-H292 cells), human bronchial epithelial cells (16HBE14o- cells), primary cultured human bronchial epithelial cells, and primary cultured human airway smooth muscle cells (positive control) (Fig. 2). These results suggest that the dopamine D₁ receptor is expressed on human airway epithelium of both trachea and bronchi as well as human airway smooth muscle.

The dopamine D₁ receptor induces production of cAMP through the stimulation of adenylyl cyclase, which is activated by the G_s protein [30]. Therefore, we examined whether activation of the dopamine D₁ receptor increases intracellular cAMP levels in human airway epithelial cells (16HBE14o- cells and NCI-H292 cells). Dopamine (1 μM) as well as dopamine D₁-like receptor agonists (SKF38393 or A68930; 1 μM) significantly increased intracellular cAMP levels in 16HBE14o- cells (dopamine; P < 0.001, SKF38393; P < 0.001, A68930; P < 0.01, n = 6) and NCI-H292 cells (dopamine; P < 0.01, SKF38393; P < 0.01, A68930; P < 0.01, n = 6) (Fig. 3a). Cyclic AMP production induced by A68930 (1 μM) was significantly reversed by pretreatment with the dopamine D₁ receptor antagonists SCH23390 (1 μM) (16HBE14o- cells: P < 0.001, n = 6; NCI-H292 cells: P < 0.05, n = 6) or SCH39166 (1 μM) (16HBE14o- cells: P < 0.001, n = 6; NCI-H292 cells: P < 0.05, n = 6) (Fig. 3b).

The increase of cAMP after dopamine D₁ receptor activation induces the activation of protein kinase A (PKA), which induces phosphorylation of CREB [14]. MEK-ERK signaling also contributes to CREB phosphorylation [13]. In addition, the dopamine D₁ receptor activates MEK-ERK signaling through β-arrestin [31]. Therefore, we examined whether the dopamine D₁ receptor agonist A68930 phosphorylates CREB through PKA and/or MEK in 16HBE14o- cells and NCI-H292 cells. A68930 (1 μM, 20 min) significantly increased phosphorylation of CREB in 16HBE14o- cells (P < 0.05, n = 3) and NCI-H292 cells (P < 0.05, n = 7) (Fig. 4a and b). The phosphorylation reached maximal levels at 20–30 min and then slowly declined to basal levels within 60 min in 16HBE14o- cells, while the increased phosphorylation was maintained at 60 min in NCI-H292 cells. To confirm that A68930 phosphorylates CREB through PKA or MEK, NCI-H292 cells were pretreated with the PKA inhibitor H89 (10 μM; 30 min) or the MEK inhibitor U0126 (5 μM; 120 min). A68930 (1 μM; 20 min)-stimulated CREB phosphorylation in NCI-H292 cells was significantly inhibited by H89 (P < 0.001, n = 4) or U0126 (P < 0.05, n = 4) (Fig. 4c). These results confirm that the dopamine D₁ receptor agonist-induced phosphorylation of CREB proceeds through both PKA and MEK/ERK signaling.

CREB was previously shown to mediate the transcriptional regulation of MUC5AC in airway epithelial cells including NCI-H292 cells [21, 22, 32]. We examined whether dopamine or the dopamine D₁ receptor agonist A68930 induces MUC5AC mRNA expression in NCI-H292 cells. Dopamine (1 μM), A68930 (1 μM), and cigarette smoke extract (CSE) (10%) significantly increased MUC5AC mRNA expression in NCI-H292 cells. The G_s protein-coupled β₂ aderenoceptor agonist isoproterenol (1 μM) also significantly induced MUC5AC mRNA expression (Fig. 5a). Previous studies in airway epithelial cells have employed final CSE concentrations ranging from 1 to 30% [33, 34]. MTT cell viability analyses confirmed that 48 h treatment of NCI-H292 cells with 10% CSE or even higher concentrations (20%) of CSE did not reduce NCI-H292 cell viability, suggesting that treatment with CSE at 10% or 20% has no cytotoxicity (Fig. 5b).

We further investigated the effect of the dopamine D₁ receptor agonist A68930 or dopamine on MUC5AC protein expression using an immunofluorescent assay. Consistent with the mRNA data of MUC5AC, MUC5AC protein expression in NCI-H292 cells were increased by dopamine (1 μM), A68930 (1 μM), and CSE (10%) (Fig. 6). These results suggest that activation of dopamine D₁ receptor stimulates MUC5AC expression.