Expression of muscarinic receptors in human and mouse sclera and their role in the regulation of scleral fibroblasts proliferation

Human sclera

Human scleral tissues (n=19) harvested within 24 h from cadaver eyes were provided by the Singapore Eye Bank. The protocol was approved by the Institutional Review Board of the Singapore Eye Research Institute and complied with the tenets of the Declaration of Helsinki.

Animals

BALB/c mice (n=102) were obtained from the animal holding unit of the National University of Singapore. All animals were housed in standard mouse cages at 25 °C on a schedule of 12 h:12 h of light on and off with mouse pellet and water available ad libitum, and the water was regularly changed. Approval was obtained from the SingHealth IACUC, and all procedures performed in this study were in accordance with the Guide for the Care and Use of Laboratory Animals and ARVO recommendations for animal experimentation.

Culture of human and mouse scleral fibroblast cells

Scleral rims from donor human cadaver eyes (n=16) and eight-week-old mouse sclera (n=150, 10 sclera/batch) from post-mortem eyes were obtained. The whole sclera was dissected very carefully and washed with cold phosphate buffered saline (PBS) three times. The human scleral tissue was cut into 2 mm×2 mm pieces, and the mouse scleral tissue was cut into 1 mm×1 mm pieces. Scleral tissues were dispersed by incubating with 0.25% trypsin/0.5 mM EDTA (Sigma, Hamburg, Germany) for 5 min at 37 °C. The tissues were placed on 60 mm×15 mm cell culture dishes (Greiner, Dresden, Germany) with a thin layer of Dulbecco’s modified Eagle’s medium (DMEM; Sigma) supplemented with 10% fetal bovine serum (FBS; Gibco BRL, San Francisco, CA), 50 U/ml penicillin, and 50 µg/ml streptomycin and amphotericin B (Sigma). Cells were grown in a humidified atmosphere of 5% CO₂ at 37 °C. After initial cell outgrowth from the explants was observed, the volume of the medium was increased. The cultures were passaged on reaching 80% confluence. All cells used in experiments were between passages 1 and 2.

Cell proliferation assay

SFs were passaged from human and mouse scleral tissues and used between passages 1 and 2. Cell proliferation was assessed by measuring 5-bromo-2-deoxyuridine (BrdU) incorporation during DNA synthesis in proliferating cells (Oncogene, Cambridge, MA). For the cell proliferation assay, 100 μl of passaged SFs (1×10⁵ cells/ml) were seeded into 96 well plates containing DMEM with 10% FBS. Cells were allowed to attach to the substratum for 24 h. The following day, the medium was replaced with fresh DMEM containing 10 µM BrdU (Oncogene) to be incorporated into newly synthesized DNA. Cells were then treated with experimental concentrations of the pan muscarinic receptor agonist, carbachol (Sigma); muscarinic toxin-1 (MT-1, a well characterized M₁-specific agonist derived from snake venom; Peptides International Inc., Louisville, KY); or the pan receptor antagonist, atropine (Sigma); or M₁ selective antagonist, pirenzepine (Sigma); muscarinic toxin-7 (MT-7, M₁ specific antagonist derived from snake venom; Peptides International Inc.); M₂/M₄ receptor antagonist, himbacine; or the M₃ receptor antagonist, 4-(diphenylacetoxy-N-methyl piperidine) methiodide (4-DAMP). To study the combined effect of atropine and carbachol, SFs were pre-treated with atropine (0.1–100 µM) 3 h before being incubated with equimolar carbachol.

After 24 h of incubation, the culture medium was removed, cells were fixed and permeabilized, and the DNA was denatured to enable antibody binding to the incorporated BrdU. Anti-BrdU antibody was added to the wells and incubated for 1 h at room temperature. Unbound antibodies were washed away, and horseradish peroxidase-conjugated goat-anti mouse was added. A substrate solution was added to each well, resulting in a color change proportional to the amount of DNA synthesis in the cells. The color reaction was stopped, and the optical density was determined using a Spectrafluor Plus microplate reader (TECAN, Durham, NC), set to 450 nm-595 nm.

Immunohistochemistry and immunocytochemistry

The whole mouse eye (two months old, n=3) and human sclera (n=3) was embedded in Optimal Cutting Temperature (OCT) compound at −20 °C for 1 h. Prepared tissue blocks were sectioned with a cryostat at 5 microns and collected on clean polysine™ glass slides (Thermo Fisher Scientific, Newington, CT). Sections were air dried at room temperature (RT) for 1 h and fixed with 4% paraformaldehyde for 10 min. After washing three times for 5 min each with 1X PBS and then adding 4% goat serum diluted with 1X PBS as a blocking buffer, slides were covered and incubated for 1 h at RT in a humidified chamber. After rinsing with 1X PBS, a specific primary antibody for M₁-M₅ (Biogenesis, Poole, England) was diluted with 2% goat serum (1:100) and incubated overnight at 4 °C in a humid chamber. After washing three times for 10 min each with 1X PBS, fluorescein-labeled goat anti-rabbit secondary antibody (1:200; Sigma) was applied and incubated for 90 min at RT. After washing and air drying, slides were mounted with an antifade medium containing DAPI (4, 6-diamidino-2-phenylindole; Vectashield; Vector Laboratories, Burlingame, CA) to visualize nuclei. For negative controls, primary antibodies were omitted.

In parallel, mouse and human SFs were cultured on sterile chamber slides. Cells were washed with PBS and fixed with ice-cold methanol:acetone (1:1) at −20 °C for 10 min and air-dried. Cells were permeabilized with 0.5% Triton X-100 in PBS for 2 min at RT. Non-specific sites were blocked with 1% bovine serum albumin (BSA), 0.3% Triton X-100, and PBS for 30 min at RT for the cells. The cells were then processed and stained as described above. The cells were mounted on coverslips with Flurosave mounting medium (Calbiochem, San Diego, CA). A fluorescence microscope (Axioplan 2; Carl Zeiss Meditec GmbH, Oberkochen, Germany) was used to examine the slides and images captured. Experiments were repeated in triplicate from three different samples.

RNA isolation and RT–PCR

The mouse globe (n=50, 10 sclera/batch) was washed with a buffered saline solution to remove traces of blood and other material. The sclera behind the optic nerve head was dissected from each eye. The whole retina including the retinal pigment epithelium (RPE) was stripped off from the sclera, and choroid was removed with a No. 10 scalpel blade. The whole sclera was then immediately frozen in liquid nitrogen.

Total RNA was extracted from human and mouse scleral tissues (TRIzol reagent; Invitrogen-Gibco, Carlsbad, CA). Genomic DNA was removed by digestion with DNase I (Amp Grade; Invitrogen-Gibco) for 15 min at RT. One microgram of total RNA was reverse-transcribed with random hexamers by using a first-strand cDNA synthesis kit (Invitrogen-Gibco). To standardize and evaluate scleral gene expression, aliquots of the same cDNA preparation were used as templates in all polymerase chain reactions (PCR) reactions. PCR amplification (PTC-200 Peltier Thermal Cycler; MJ Research, Ramsey, MN) was performed using Taq DNA polymerase (Promega, Madison, WI). Each reaction confirmed that the PCR products were not saturated. The primers for muscarinic receptors were constructed with the Primer Express Software version 1.0 (PE Applied Biosystems, Foster City, CA) using a mouse and human specific acetylcholine muscarinic receptor subtypes sequence (Table 1 and Table 2, respectively). Total RNA extracted from the mouse brain was used as a positive control. PCR amplification of β-actin was performed in parallel as an internal control to detect genomic DNA contamination. The amplified products were analyzed by electrophoresis on 1.2% and 2.5% agarose-TAE gels (human and mouse sclera, respectively) and photographed under ultraviolet (UV) light. All experiments were done in triplicate.

Relative and absolute quantitative real-time PCR

Real-time PCR primers for transcripts of M₁-M₅ were purchased from Applied Biosystems Inc. (Taqman Gene Expression System; ABI, Foster City, CA), and Quantum RNA classic II 18S Internal Standard (Ambion, Austin, TX) was used as an endogenous control. The 18S primer-competimer ratio of 1:4 was used in all experiments. Real-time PCR reactions were performed on a sequence detection system (Prism 7700; ABI) with 500 ng of total cDNA per reaction in a final volume of 25 µl. The C_T (threshold cycle) of each reaction was obtained by using a constant threshold with the 18S rRNA used as an internal control. ∆C_T was calculated by subtracting the average C_T of 18S rRNA from the average C_T of the target gene. All experiments were performed in triplicate. This was repeated with five different batches of samples. The conditions for the PCR were as follows: 55 °C for 2 min, 95 °C for 10 min and 40 cycles each of 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 2 min. The comparative quantification values were obtained from the threshold cycle (C_T) number at which the increase in signal was associated with an exponential growth of PCR products and this was detected in SDS (Satellite Data System) software in ABI PRISM® 7700 Sequence Detection System. The PCRs were performed (n=5) for each sample-primer set, and the mean of the experiments was used as the relative quantification value. For all M₁-M₅ probes, the ABI-confirmed amplification specificity of TaqMan probes and the reaction products were separated on 3% agarose gel, stained with ethidium bromide for visual confirmation of PCR products, and sequenced.

Data analysis by comparative C_T method

The C_T value represented the PCR cycle at which an increase in reporter fluorescence (∆Rn) above the line of the optimal value (optimal ∆Rn) was first detected. The calculation for the comparative C_T (∆∆C_T) method was previously described [32]. 18S rRNA was used as an endogenous internal control. The average C_T value of all muscarinic receptor genes and 18S rRNA was calculated from each experiment of a triplicate, and this was used for all five samples. Finally, the mean C_T value was calculated from the five average C_T values. The ∆C_T value was determined by subtracting the corresponding mean 18S rRNA C_T value from the mean of the M₁-M₅ C_T value. The standard deviation of the difference was calculated from the standard deviations of the gene of interest and the corresponding 18S rRNA values. The ∆∆C_T value was obtained by subtracting the ∆C_T calibrator value. This was the subtraction of an arbitrary constant so the standard deviation of the ∆∆C_T was similar to the standard deviation of the ∆C_T value. The expression level of each gene in the mouse brain was used for calibration. ∆∆C_T of the scleral sample was calculated by subtracting the ∆C_T of the mouse brain from ∆C_T of the scleral sample. The relative change of scleral samples compared with brain samples was determined as 2^-∆∆CT. Statistical analysis was performed by ANOVA. The expression levels of the genes for M₁–M₅ in the sclera were compared by the Fisher LSD (Least Significant Difference) test. A probability level of p<0.05 was considered statistically significant.

Phospho-p42/44 MAPK activation assay

Mouse SF cell lysate was exposed to atropine and carbachol at 0, 0.1, 1, 10, and 100 µM for 0.5 h. Phospho-p42/44 mitogen-activated protein kinase (MAPK) assay kit were purchased from Assay Design (Ann Arbor, MI), and all procedures were conducted according to the instructions provided by the company. A monoclonal antibody specific for phospho-p42/44 MAPK was pre-coated onto a 96 well microtiter plate. Standards and samples were added into the wells, and p42/44 MAPK became bound to the immobilized antibody. Proteins were removed by washing, and an enzyme-linked monoclonal antibody specific for p42/44 MAPK was added to the wells. Washed again to remove any unbound antibody-enzyme reagent and the calorimetric, a substrate solution was added to the wells. Color was developed in proportion to the amount of p42/44 MAPK bound in the initial step. The intensity of the color was measured at 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader (TECAN, Durham, NC) to determine the levels of phosphorylated p42/44 MAPK proteins.

Western blotting-protein expression of mAChRs

The nearly confluent mouse and human SFs were homogenized in ice-cold radio immunoprecipitation assay (RIPA) buffer containing proteinase inhibitors (10 mM Tris-HCl [pH 7.4], containing 150 mM NaCl, 1% deoxycholic acid, 1% Triton X-100, 0.1% SDS, 1 mM EDTA, 10 mg/ml phenylmethylsulfonyl fluoride, 5 U/ml aprotinin, and 100 nM sodium orthovanadate) and phosphatase inhibitors (Pierce, Rockford, IL). After homogenization, the samples were centrifuged at 14,000x g for 10 min at 4 °C, and the supernatants were used as total cell lysates.

Total protein concentration was determined using a direct colorimetric (DC) protein assay reagent (Bio-Rad, Hercules, CA). Theses proteins were used to detect the muscarinic receptors in both human and mouse SF using immunoblot. Proteins in the supernatant were separated by SDS–PAGE, transferred to nitrocellulose membranes, blocked in 5% BSA in TBST (10 mM Tris-HCl [pH 8.0], 150 mM NaCl, and 0.05% Tween-20) for 2 h at RT, and incubated with the same anti-muscarinic receptor antibodies described earlier at a dilution of 1:1000 to detect the mAChRs proteins.

Analysis of Erk1/2 and Ras phosphorylation by carbachol and FGF-2 on mouse SF

The mouse SF cells were cultured in a serum-containing medium in six wells (2×10⁵ cells/well). Cultured mouse SFs were starved for 24 hours and then was incubated with 50 mg/ml carbachol or 50 ng/ml FGF-2 for 0.5, 1, 2, 6 h and over night to detect the optimal time required for MAPK (ERK1/2) and Ras phosphorylation in these cells. For detection, western blot was performed as described below.

Analysis of phosphorylation by muscarinic agents in mouse SF

The mouse SF cells were cultured in a serum-containing medium in six wells (2×10⁵ cells/well). From the earlier analysis, we found that 30 min was the optimal time to detect the ERK1/2 phosphorylation. Hence, we have treated the mouse SFs with freshly prepared atropine, pirenzepine, and carbachol at a concentration of 50 µM and MT-1 and MT-7 at a concentration of 0.5 µM for 30 min to determine the signaling phosphorylation of EGF-R, PKC, Pyk2, B-Raf, Ras, JNK1/2 and ERK1/2 proteins with the effect of muscarinic agents. Protein extraction and electrophoresis were performed as described above.

For detection, the membrane was incubated with anti-Ras clone RAS10, phospho-EGF-R, PKC, Pyk2, B-Raf, Ras, JNK1/2, and ERK1/2 antibodies at the dilution of 1:1000 according to the instructions provided by the company (Millipore, Billerica, MA) for 2 h at RT, and β-tubulin was used as a loading control and was incubated for 1 h at RT.

All three experiments’ membranes were washed three times in TBST and incubated with HRP-conjugated secondary antibody (Chemicon International, Temecula, CA) at a dilution of 1:2500 for 1 h at RT. Immunoreactive bands were visualized using the enhanced chemiluminescence method (GE Healthcare, Buckinghamshire, UK). The membrane was wrapped in plastic and placed against X-ray film, exposed for an appropriate length of time (30 s to 5 min), and developed (Kodak, Rockford, IL).

Growth factor receptors ELISA

Mouse SF cell lysate was exposed to atropine and carbachol at concentrations of 0, 0.1, 1, 10, and 100 µM for 24 h. Cellular EGF, fibroblast growth factor-2 (FGF-2), and transforming growth factor (TGF)-β1 receptors in the presence of muscarinic agents was quantified by an ELISA. Equal amounts of samples or standards were added to each well (anti-EGF-R, -FGF-2, and -TGF-β1 antibody pre-coated) and incubated for 2 h at RT. The wells were aspirated and washed with PBST. Anti-EGF, -FGF-2 (Chemicon International Inc.), and -TGF-β1 (Amersham Pharmacia Biotech, Buckinghamshire, UK) mouse IgG conjugated with horse-radish peroxidase was added to the respective wells and incubated for 2 h at RT. Substrate solution was added to each well, resulting in a color change proportional to the amount of EGF-R, FGF-2, and TGF-β1 present in the samples. Samples were allowed to develop color for 20 min at RT. Adding stop solution stopped the reaction, and the optical density was determined within 30 min using Spectrafluor Plus microplate reader (TECAN) at dual wavelengths of 450 nm and 620 nm. Quantification was achieved by the construction of a standard curve using the known concentration of EGF, FGF-2, and TGF-β1 receptors.

Data analysis

Statistical comparisons between experimental groups were conducted using Student’s t-test or one-way ANOVA (Statistica 6.0; SPSS, Chicago, IL) followed by Tukey post hoc test. A significance level of p<0.05 was used. Data are presented as means±SEM (standard error of the mean).

Cellular expression of M₁-M₅

Immunohistochemical localization of muscarinic receptors M₁-M₅ was performed in passage 2-cultured human and mouse scleral fibroblasts (SFs) as well as scleral tissues. Positive immunostaining for all five mAChR subtypes was shown in the cultured human and mouse SFs (Figure 1A,B, respectively) as well as in human and mouse scleral tissues (Figure 2A,B, respectively). The M₁-M₅ receptors were localized to the cell membrane as well as to the cytoplasm. No immunostaining was observed in the negative controls. Thus, all five receptor types were represented in mouse and human scleral fibroblasts at passage 2 as well as in the mouse scleral tissue.

Gene expression of muscarinic receptor subtypes

RNA (1 µg) sample from each collected tissue (n=10 sclera) was separated on a 1.2% agarose gel to determine the RNA purity before further analysis. By conventional PCR, expression of all five mAChRs was detected in human and mouse sclera (Figure 3A,B, respectively). The identity of each PCR product was further confirmed by sequencing. Mouse primer sequences showed a high sequence identity to the NCBI Blast mAChR subtypes of mouse sequences (M₁ 99%, M₂ 100%, M₃ 100%, M₄ 100%, M₅ 100%). As none of the five mAChR genes has introns, the PCR product from genomic DNA would yield the same-sized amplicons. To rule out possible contamination by genomic DNA, we chose a pair of β-actin primers at two adjacent exons that spanned a 206 bp intron on cDNA derived from β-actin mRNA. The identification of a single reverse transcription polymerase chain reaction (RT–PCR) product of predicted size for β-actin mRNA rules out contamination by genomic DNA. The β-actin primers were designed to span a 206 bp intron, finding a single band of 314 bp verifies that genomic DNA was not present.

Quantitative real-time PCR was used to compare the relative abundance of the transcript of each mAChR in the sclera. These levels were also compared to M₁-M₅ gene expression in the mouse brain cerebellum. Normalized expression levels of M₁, M₃, and M₄ were lower in the sclera when compared to the levels for the mouse cerebellum (p<0.001). However, the levels of M₂ and M₅ were greater in the sclera when compared to the mouse cerebellum (p<0.05, n=10 sclera from five mice; Figure 4).

Western blot analysis

Western blot analysis using SFs at passage 3 was performed using human and mouse cells to determine levels of protein expression of each muscarinic receptor subtype. Figure 5 shows that major bands representing M₁ (58 kDa), M₂ (52 kDa), M₃ (52 kDa), M₄ (66 kDa), and M₅ (65 kDa) were detected. All five mAChR proteins were expressed in both mouse (Figure 5A) and human (Figure 5B) SFs.

Muscarinic antagonists inhibited DNA synthesis

As muscarinic receptors are often linked to proliferation, we used this as an indicator of cell function of pharmacological control exerted through mAChRs. SFs were exposed to atropine, pirenzepine, or MT-7 for 24 h using a range of concentrations. By microscopic observation, cell morphology showed little differences between treatment groups and control groups, and viability as determined by the trypan blue exclusion assay showed that there was no toxicity after 24 h incubation of the cells with up to 100 μM of atropine (EC₅₀=501.1 μm or 0.5011 mM) and pirenzepine (EC₅₀=7040 μm or 7.040 mM) or 1 μM of MT-7. DNA synthesis, measured after 24 h using a BrdU ELISA assay, was inhibited after atropine, pirenzepine, and MT-7 exposure when compared with untreated control SFs (p<0.05, n=4; Figure 6A,B). In addition, when examining the competition between the agonists and antagonists, atropine, pirenzepine, and MT-7 blocked the effect of the action of carbachol (EC₅₀=177.827 mM) and MT-1 (muscarinic agonist) when SFs were pre-treated (3 h) with an equimolar amount of antagonists (Figure 7). MT-7, which is selective for the M₁, was almost as effective as atropine, a pan-muscarinic blocker, in decreasing BrdU uptake.

Effect of muscarinic agents on MAPK activity

From the preceding data, it would be suggested that the MAPK pathways should be activated. We examined phosphorylation of p42/44 MAPK in mouse SFs following exposure to atropine, pirenzepine, and carbachol at concentrations of 0.1, 1, 10, and 100 µM and to MT-7 and MT-1 at concentrations of 0.001, 0.01, 0.1, and 1 µM using ELISA. Phospho-p42/44 MAPK was formed in a dose-dependent manner (Figure 8). Phospho-p42/44 MAPK decreased with the muscarinic antagonists and increased in response to the agonists (p<0.05, n=4).

Immunoblot analysis

The muscarinic receptors have been involved in trans-activation with tyrosine kinase receptors. We examined several specific points in the pathway to provide further information about the MAPK pathway. Phospho-EGF-R, -PKC, -Pyk2, -B-Raf, -Ras, -JNK1/2, and total/phospho-Erk1/2 were expressed at protein levels in mouse scleral fibroblasts following 30 min exposure to muscarinic agents, and β-tubulin was used as the loading control (Figure 9). The antagonists (atropine, pirenzepine, and MT-7) inhibited the formation of phospho-EGF-R, -PKC, -Pyk2, -B-Raf, -Ras, -JNK1/2, and -ERK1/2. In the absence of treatment, there was no effect on total Erk1/2, β-tubulin, or control. In contrast, in SFs that were treated with the agonists (carbachol and MT-1), the activation of phospho-EGF-R, -PKC, -Pyk2, -B-Raf, -Ras, -JNK1/2, and -ERK1/2 increased.

Carbachol-induced Ras and ERK1/2 phosphorylation

We further demonstrated the carbachol-Induced Ras and ERK1/2 phosphorylation in mouse SFs to show the muscarinic agents also had parallel effects on the appropriate signal transduction pathways. As seen below, carbachol produced a time-and dose-dependent increase in Ras and MAPK/ERK phosphorylation in SFs.

We examined the activation of Ras and p42/44 MAPK in the muscarinic receptor-mediated responses in SF cells. Stimulation with 50 mg/ml of carbachol resulted in increased activity in Ras and p42/44 MAPK in a time-dependent manner (Figure 10). Ras and p42/44 MAPK activation was initially observed at 30 min with 50 mg/ml of carbachol stimulation (p<0.05, n=4). As a comparison to the activation of p42/44 kinase elicited by carbachol, we tested fibroblast growth factor (FGF-2) at 50 ng/ml, which was also found to activate Ras and p42/44 MAPK (Figure 11) with peak phosphorylation at 30 min (p<0.05, n=4). At 2 h, the level of Ras and phospho-p42/44 MAPK returned to basal levels (p<0.05, n=4). The p42/44 MAPK activation patterns for carbachol and FGF-2 were similar (Figure 10 and Figure 11).

Carbachol stimulates EGF-R and TGF-β1 activity

Past study illustrated that scleral cells are directly responsible for DNA synthesis and extracellular matrix synthesis that produces axial elongation [33]. Muscarinic agents may act directly on sclera fibroblast through the G protein coupled muscarinic receptor to modulate postnatal eye development. We assume that the effect of muscarinic antagonists may also be mediated directly or indirectly by growth factors through receptor tyrosine kinases, which would then control sclera fibroblast activity. For these reasons, we focused on examining the activation of EGF receptor and TGF-β1 in the muscarinic receptor-mediated responses in SF cells. On stimulation with 0.1, 1, 10, and 100 µM concentrations of carbachol, EGF receptor, and TGF- β1 activities (Figure 12A,B, respectively) were increased in a dose-dependent manner. On stimulation with 0.1, 1, 10, and 100 µM concentrations of atropine, the EGF receptor and TGF-β1 activities were inhibited in a dose-dependent manner (p<0.05, n=4).

Muscarinic antagonists modulates fibroblast growth factor-2 (FGF-2) activity

In this experiment we showed that muscarinic agents modulate fibroblast growth factor expression in these cells. Atropine increased FGF-2 activation in a dose-dependent manner. In contrast, carbachol decreased FGF-2 stimulation in a dose-dependent manner (Figure 13A,B). Intraocular injection of FGF-2 can prevent axial elongation induced by form deprivation [34], suggesting that form deprivation might be associated with a reduction of FGF-2 or its receptors.