Serum Deprivation Can Suppress Receptor-Mediated Calcium Signaling in Pterygial-Derived Fibroblasts

Abstract

Purpose.: Pterygium is characterized as invasive, proliferative fibrovascular altered conjunctival tissue. The extensive vascular network is likely to significantly contribute to the progression of the disease. In the present study, we investigated the effects of reduced serum (to mimic a suppressed blood supply) on cell signaling events and the functional role of the calcium store in cultured, pterygial-derived fibroblasts.

Methods.: Pure fibroblast cultures were established from cell outgrowths of pterygial tissue. Growth and migration of pterygial-derived fibroblast was evaluated using a patch growth assay, MTS assay, and a scratch wound assay. Intracellular calcium levels were determined using Fura-2 detection in response to ligand stimulation using a 96-well plate format.

Results.: A progressive increase in serum concentration resulted in promotion of pterygial cell growth detected using the MTS assay. A significant increase in intracellular calcium level was observed in response to histamine (10 and 100 μM), ATP (10 and 100 μM), acetylcholine (10 and 100 μM) and epidermal growth factor (10 ng/mL) in serum-maintained cells. However, no significant changes were observed when cells were maintained in serum-free medium. Thapsigargin (1 μM), a Ca-ATPase inhibitor, induced a significantly greater increase in intracellular calcium level in the serum-maintained group relative to serum-starved cells. In both cases, elevation of intracellular calcium was reduced when calcium-free bathing medium was used. Preincubation of cells with 1 μM thapsigargin ablated ligand-induced calcium responses. Disruption of calcium signaling through thapsigargin treatment significantly perturbed cell growth and migration.

Conclusions.: Receptor-induced calcium signaling activity is suppressed in pterygial-derived fibroblasts in response to serum deprivation. This correlates with reduced growth rates and a depleted endoplasmic reticulum calcium store. The store plays a key role in cell growth and migration of pterygial-derived fibroblasts. Therefore, the strategic reduction of the vascular network in pterygium will affect the calcium store level and in turn affect functional responses associated with pterygia.

Introduction

Pterygium is an ocular surface disease, characterized as invasive, proliferative fibrovascular-altered conjunctival tissue and fleshy outgrowth over the cornea. It is triangular in shape (wing shape appearance) and grows most commonly from the nasal aspect of the sclera, proliferating on the naso-temporal axis. In addition, it occurs more commonly in outdoor workers (most commonly in tropical and equatorial regions), such as fishermen and farmers who experience long-term irritant stimuli from exposure to sunshine and dust. Pterygia cause inflammation, irritation, and may affect vision by inducing astigmatism or involvement of the visual axis. Although the pathogenesis of pterygia is unclear, the photobiology effect of ultraviolet (UV) exposure leading to limbal epithelial cell damage is commonly accepted. ^1,2 In theory, it suggests that pterygium is the result of subconjunctival invasion of fibroblasts that enter the cornea along natural tissue planes surrounding Bowman's layer, ³ accompanied by stromal ingrowth of capillaries in the epithelium of pterygia. ⁴ Additionally, inflammatory cell infiltrate and overexpression of extracellular matrix with alteration of the collagen and elastic fibers are also contributing factors. ^2,3,5 However, pterygial fibroblasts show a greater growth response and release of growth factors compared with normal conjunctival fibroblasts under the same conditions. ^6,7 Transdifferentiation is an essential process in the genesis of fibroblasts in human tissues, embryogenesis in vertebrates, ⁸ and forming tissue fibrosis. ⁹ It is reported that the fibrogenic stimuli may induce transdifferentiation from fibroblasts to myofibroblasts, which is reported in fibrovascular tissues of primary and recurrent pterygia. ^10,11 Moreover, pterygium is characterized as a highly vascular tissue. It is reported that VEGF is highly expressed and more von Willebrand factor (vWF) is stained in new vessels in pterygium tissue compared with normal conjunctiva. ¹² Therefore, angiogenesis is likely to play a role in pterygium. ^12,13 It has been proposed that the abundance of proangiogenic factors in pterygium may irritate the limbal basal cells, produce vessel ingrowth, and promote formation of pterygium. ¹  

Calcium is an essential element in maintaining normal physiological function. It is considered a versatile second messenger that is able to regulate many cellular physiology processes. ¹⁴ Many of the physiological features of pterygium such as persistent cellular proliferation, transdifferentiation, and angiogenesis could be attributed to Ca²⁺ signaling activities. ² The regulation of calcium signaling through recruitment of the endoplasmic reticulum store has not been well studied in pterygial cells. The present study therefore aimed to understand the effects of serum deprivation on calcium cell signaling events and the functional role of calcium signaling per se in pterygial-derived fibroblasts. 

Methods

Cell and Tissue Preparation

Pterygia were harvested at routine operation with full Research Ethics Committee approval and were used in accordance with the tenets of the Declaration of Helsinki. Pterygia were placed directly into an Eppendorf tube containing Eagle's minimum essential medium (EMEM; Sigma-Aldrich, Poole, Dorset, UK) supplemented with 10% fetal calf serum (FCS) and 50 μg/mL⁻¹ gentamicin. Pterygium was then either fixed in 4% formaldehyde for immunohistochemical analysis or transferred to a 35-mm tissue culture dish and secured using entomological pins (D1; Watkins and Doncaster Ltd., Cranbrook, Kent, UK) and cultured as previously described by Maini et al. ¹⁵ Following a period of culture in 10% FCS supplemented EMEM at 35°C in a 5% CO₂ atmosphere, pterygia were transferred to a new dish and maintained under the same culture conditions until cellular outgrowths were fibroblast only. Characterization of all cultures is routinely performed using immunocytochemistry for fibroblast and epithelial cell markers. 

Measurements of Intracellular Calcium Levels

Pterygial fibroblasts were seeded in each well of a transparent 96-well microtiter plate (NUNC 96-well plate; Fisher Scientific, Loughborough, UK) at 5000 cells in 200 μL 10% FCS-EMEM and allowed to establish. Medium was removed and replaced with serum free EMEM or EMEM supplemented with 10% serum for a 48-hour period. The cells were then loaded with 5 μM Fura 2-acetoxymethylester (Fura 2-AM) for 40 minutes. Following this period, the medium in each well was changed to standard Ringer's solution (AAH; 5 mM KCl, 5 mM NaHCO₃, 5 mM glucose, 20 mM HEPES, 130 mM NaCl, 0.5 mM MgCl₂, 1 mM CaCl₂, pH 7.25) or calcium-free Ringer's solution (AAH; 5 mM KCl, 5 mM NaHCO₃, 5 mM glucose, 20 mM HEPES, 130 mM NaCl, 0.5 mM MgCl₂, 1 mM EGTA, pH 7.25). ¹⁶ The plate was placed in a BMG Labtech Fluostar Omega plate reader (BMG LABTECH GmbH, Offenburg, Germany), which was maintained at 35°C. Fura 2 was excited alternately with 340- and 380-nm wavelengths with emission detected at 520 nm. The results were analyzed by control and analysis software (MARS Data Analysis; BMG LABTECH GmbH). Calcium levels were initially determined by the ratio of Fura-2 signal at 340 nm/signal at 380 nm. The ratio value at the onset of the experiment (t = 0) served as a reference signal (F0); all subsequent readings were established as an F1/F0 ratio. Peak responses following stimulation were determined using the MAX feature on the commercial spreadsheet software used (Excel; Microsoft Corp., Redmond, WA). 

Patch Growth Assay

Pterygial fibroblasts were seeded on a 35-mm tissue culture dish at 5000 cells in 200 μL 10% FCS-EMEM and allowed to establish over a 48-hour period, such that a distinct patch of cells (∼1 cm in diameter) was observed. The medium was then removed and replaced with serum-free EMEM for a 24-hour period. The medium was again removed and placed into experimental conditions for 48 hours; conditions were 10% FCS EMEM ± 1 μM thapsigargin. At endpoint, cells were washed with PBS and stained with Coomassie blue (Merck KGaA, Darmstadt, Germany) for 1 hour to enable the patches to be visualized and measured. The dye was removed and the cells were washed with PBS several times to remove excess dye. Images of individual patches were captured on a CCD camera using image acquisition software (GeneSnap; Syngene, Cambridge, UK). Cell coverage was determined using ImageJ software (National Institutes of Health [NIH], Bethesda, MD). To provide an estimate of total cell number, the Coomassie blue dye within the cells was extracted by removal of PBS and addition of 1 mL 70% ethanol. 100 μL samples of each dish were transferred to a 96-well plate (NUNC 96-well plate; Fisher Scientific). The level of dye content was assessed by measuring absorbance at 550 nm using a BMG Labtech Fluostar Omega plate reader (BMG LABTECH GmbH). 

MTS Assay

Pterygial fibroblasts were seeded in each well of a transparent 96-well microtiter plate (NUNC 96-well plate; Fisher Scientific) at 5000 cells in 200 μL 10% FCS-EMEM and allowed to establish. The medium was removed and replaced with serum free EMEM for a 24-hour period. The medium was then removed and placed into experimental conditions for 24 or 48 hours. The pterygial fibroblast cell number was determined using a commercial cell viability assay (CellTiter 96 AQ_ueous Non-Radioactive Cell Proliferation Assay; Promega, Madison, WI). This is a colorimetric method for determining the number of viable cells in proliferation, cytotoxicity or chemosensitivity assays based on the reduction of MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H- tetrazolium, inner salt]. The plate was read at 490 nm using a BMG Labtech Fluostar Omega plate reader (BMG LABTECH GmbH). 

Scratch Wound Assay

Pterygial fibroblasts were seeded on a 35-mm tissue culture dish at 5000 cells in 200 μL 5% FCS-EMEM and allowed to establish over a 48 hour period, such that a distinct patch of cells (∼1 cm in diameter) was observed. The medium was then replaced with nonsupplemented EMEM and cultured for an additional 24 hours. At this time point, a scratch was made through the middle of the confluent sheet using a plastic pipette tip. Indentations within the wound area were made to establish points of reference. The cells were then exposed to experimental conditions and maintained for 24 hours with the medium being changed every 2 days. Images were captured and the movement determined using image analysis software (ImageJ; NIH). 

Results

Concentration-Dependent Effects of Serum on Cell Growth

A concentration-dependent effect of serum over a 48-hour culture period was observed with pterygial fibroblasts (Fig. 1). Concentrations of 4% FCS and above were required to produce a significant increase in growth. A peak response was obtained with 10% FCS addition, such that the cell population was 183 ± 11.67% relative to nonstimulated serum-free control. 

Figure 1. 

 

Concentration-dependent effects of serum on cell growth of pterygial-derived fibroblasts detected using the MTS assay. The data represent mean ± SEM pooled from experiments performed on pterygial-derived fibroblasts from three different donors. Cells were exposed to experimental conditions for a 48-hour culture period. *Indicates a significant difference between nonstimulated control and treated group (P ≤ 0.05; ANOVA with Dunnett's post hoc test).

Calcium Signaling

To assess putative changes in calcium signaling in serum-maintained and serum-free conditions, we employed several agonists that are known to mobilize intracellular calcium. These ligands were histamine, ATP, ACh, and EGF. Moreover, these ligands were selected because they have been associated with pterygia or have the potential to influence this condition. For example, active histamine and epidermal growth factor receptors have been determined in human pterygia. ¹⁷ Extracellular ATP levels are also known to rise in stressed or pathological states, ¹⁸ and ACh is believe to influence cell function as a consequence of innervation, but can also transmit signals independent of innervation. ¹⁹  

In serum-maintained cells, histamine, ATP, ACh, and EGF were shown to increase intracellular calcium levels through mobilization of the ER calcium store in a concentration-dependent manner. Significant elevation of intracellular calcium was observed with concentrations ≥ 10 μM histamine, ATP, and ACh and 10 ng/mL in the case of EGF. In contrast, cells maintained in serum-free medium and exposed to the four ligands did not show any significant increase in intracellular calcium levels at any of the concentrations tested. Responses obtained with histamine, ATP, ACh at 100 μM and EGF at 10 ng/mL differed significantly between the serum maintained and serum-starved cells (Fig. 2). Moreover, thapsigargin was applied to deplete the calcium store in pterygial fibroblasts in the presence and absence of serum and thus reveal the level of calcium stored within the ER. Thapsigargin (1 μM) induced a significant increase in intracellular calcium level of cells maintained in serum and serum-starved conditions (Fig. 3). However, a significantly greater increase in calcium was observed in the serum-maintained cells; the peak intracellular calcium level in serum-free maintained cells group was 47% that obtained in serum-maintained cells (Fig. 3). To assess the role of calcium influx, through store operated calcium entry, in the thapsigargin elevation of intracellular calcium experiments were performed in calcium-free extracellular medium (Fig. 3). These experiments demonstrated an overall reduction in intracellular calcium levels in response to 1-μM thapsigargin, but peak responses were not significantly different from cells treated in the presence of calcium in the extracellular medium. 

Figure 2. 

 

Characterization of ligand-mediated calcium mobilization in pterygial-derived fibroblasts cultured in the presence and absence of serum. Baseline calcium levels were established prior to injection of agonists after 15 seconds as indicated; the ligand was retained in the bathing medium for the remainder of the experiment. The data represent mean ± SEM pooled from experiments performed on pterygial-derived fibroblasts from three different donors. *Indicates a significant difference from baseline; ΔSignificant difference between serum-maintained and serum-starved groups (ANOVA with Tukey's post hoc test, P ≤ 0.05).

Figure 3

 

The effect of serum starvation on ER calcium store depletion in response to the Ca-ATPase inhibitor thapsigargin in the presence and absence of extracellular calcium. Baseline calcium levels were established prior to injection of 1 μM thapsigargin after 15 seconds as indicated; the ligand was retained in the bathing medium for the remainder of the experiment. The data represent mean ± SEM pooled from experiments performed on pterygial-derived fibroblasts from four different donors. *Indicates a significant difference from baseline. ΔSignificant difference between serum-maintained and serum-starved groups (Student's t-test, P ≤ 0.05).

In order to confirm that the ligand-induced calcium responses involved the ER calcium store, we pretreated cells with thapsigargin for >1 hour to ensure the calcium store was drained. Control cells maintained in serum-free or serum, that exhibit a normal ER store, increased intracellular calcium in response to all ligands with a greater response observed in serum-maintained cells (Fig. 4). In all cases, cells pretreated with thapsigargin did not elicit an elevation in calcium in response to any of the four ligands tested. 

Figure 4. 

 

The effect of ER calcium store depletion by thapsigargin on ligand-mediated calcium responses. Cells were maintained in control medium or exposed to 1 μM thapsigargin for the final hour of culture prior to Fura-2 loading. Baseline calcium levels were established prior to injection of agonists after 15 seconds as indicated; the ligand was retained in the bathing medium for the remainder of the experiment. The data represent mean ± SEM pooled from experiments performed on pterygial-derived fibroblasts from three different donors. *Indicates a significant difference from baseline. ΔSignificant difference between serum-maintained and serum-starved groups (ANOVA with Tukey's post hoc test, P ≤ 0.05).

The Effects of Calcium-Signaling Disruption on Cell Growth and Migration

To determine the importance of calcium signaling on pterygial cell function, we employed several assays to evaluate growth and migration. A patch assay was initially used to assess coverage of cells on a tissue culture dish. Thapsigargin (1 μM) was used to disrupt calcium signaling. Thapsigargin treated cells covered a significantly smaller area, such that area covered was 65.3% ± 5.39% that of the control group (Fig. 5). Cell population numbers also differed significantly. In this case, thapsigargin-treated cells were 86.3% ± 1.6% compared with controls (Fig. 5). These data suggested an effect on both migration and cell growth. We then expanded on these findings and employed an MTS assay to further evaluate effects on cell populations and a scratch assay to study effects on migration. The MTS assay showed a similar outcome to the patch assay, such that populations in the thapsigargin-treated group were 81.7% ± 3.1% (Fig. 6). The scratch wound assay clearly demonstrated the ability of thapsigargin to inhibit coverage of the wounded area (Fig. 7). Within 24 hours, control cells have rapidly covered the cell-free area, whereas progress is significantly retarded in the thapsigargin-treated group. 

Figure 5. 

 

The effect of thapsigargin on pterygial-derived fibroblast growth detected using the patch assay. Pterygial fibroblasts were cultured for 48 hours in EMEM supplemented with 10% FCS ± 1 μM thapsigargin. (A, B) Representative images of patch assays maintained in the absence (A) and presence (B) of thapsigargin. (C, D) Pooled quantitative data from experiments performed on pterygial-derived fibroblasts from three different donors showing changes in cell coverage (C) and cell population (D). The data represent mean ± SEM. *Indicates a significant difference between nonstimulated control and treated group (P ≤ 0.05; Student's t-test).

Figure 6. 

 

Effects of thapsigargin on cell growth of pterygial-derived fibroblasts detected using the MTS assay. Pterygial fibroblasts were cultured for 24 hours in EMEM supplemented with 10% FCS ± 1 μM thapsigargin. The data represent mean ± SEM pooled from experiments performed on pterygial-derived fibroblasts from three different donors. *Indicates a significant difference between nonstimulated control and treated group (P ≤ 0.05; Student's t-test).

Figure 7. 

 

Effects of thapsigargin on cell migration of pterygial-derived fibroblasts detected using the scratch assay. Pterygial fibroblasts were cultured for 24 hours in EMEM supplemented with 10% FCS ± 1 μM thapsigargin. (A) Representative images showing the scratch wound at t = 0 and endpoint. (B) Pooled quantitative data from experiments performed on pterygial-derived fibroblasts from three different donors. The data represent mean ± SEM. *Indicates a significant difference between nonstimulated control and treated group (P ≤ 0.05; Student's t-test).

Discussion

A number of techniques for treatment of pterygium are available. Current therapeutic approaches include conjunctival autograft, sliding conjunctival flaps, excimer laser treatment, and amniotic membrane transplants. ²⁰ In addition, the adjunctive therapies such as radiation therapy, ²¹ intraoperative, and postoperative mitomycin, ^22,23 can be employed to prevent recurrence of pterygia. It is reported that pterygia exhibit a high degree of vasculature relative to normal conjunctiva, which has a relatively limited vasculature. ²⁴ The proangiogenic VEGF has been identified in pterygium specimens and its presence links in well with the abundance of blood vessels. ²⁵ Therapeutic approaches using anti-VEGF have been used and some benefit has been reported. ²⁶ It was therefore of interest to determine the importance of this rich vasculature and determine the impact of restricting nutrient supplies to pterygial cells. A simple approach was used, which involved serum maintenance or serum starvation. The intention of this strategy was to mimic an environment that has a rich blood supply and one that is poor. As predicted, a progressive decrease in cell growth was observed as serum levels declined. Pterygial cells are therefore reliant on a supply of growth and survival factors. Having established that serum starvation reduces cell growth, analysis of calcium signaling was performed. 

Calcium is considered a major and versatile second messenger in virtually every cell to regulate key cellular physiology processes, such as gene expression, proliferation, contraction, and cell metabolism. ²⁷ Pterygial tissue is characterized by extensive cellular proliferation, transdifferentiation, and angiogenesis, which is linked to Ca²⁺-signaling activities. ² The endoplasmic reticulum (ER) is the main intracellular Ca²⁺ store/release organelle. The cells can either elevate cytosolic Ca²⁺ by releasing Ca²⁺ from intracellular store or uptake Ca²⁺ into the cell from extracellular solution. Store-operated calcium entry (SOCE) is the dominant Ca²⁺ entry pathway. ²⁸ Two major receptor-mediated pathways are involved in the formation of inositol trisphosphate (IP₃) to release Ca²⁺ from ER. G-protein–coupled receptors including histaminergic, purinergic, and muscarinic receptors activate one pathway by stimulation of PLCβ. Alternatively, EGF is a tyrosine kinase-linked receptor that can utilize PLCgamma to elevate IP₃ and mobilize the ER calcium store. ²⁷ Histamine, ATP, acetylcholine, and epidermal growth factor were evaluated for their effect on intracellular calcium levels of pterygial fibroblasts in the presence and absence of serum. All ligands demonstrated a reduced ability to raise intracellular calcium concentration following serum starvation. This suggests that signaling is impaired and may be the result of differences in receptor expression or modification of signaling molecules common to these ligand/receptor systems. However, all ligands show a similar pattern of response, which suggests that a common factor is changing. To address this issue, we concentrated our efforts on the calcium store itself. The Ca-ATPase inhibitor thapsigargin can be used to establish store content. It depletes the ER store slowly and evokes the cytosolic free Ca²⁺ without elevation in inositol polyphosphates. ²⁸ Application of thapsigargin to cells maintained in serum-free medium demonstrated that the calcium level within the endoplasmic reticulum store was depleted relative to serum-maintained cells. Therefore, these data illustrate that intracellular calcium stores are sensitive to serum deprivation: this could have a marked effect on cell behavior. The ER calcium store is regulated by a number of components. The receptor channels inositol-1, 4, 5-phosphtate- (IP₃R) and ryanodine-receptors (RYR) release Ca²⁺ from intracellular stores. ²⁹ Therefore, reduction in these channels would impair release from the store. In addition, sarcoplasmic/endoplasmic-reticulum-Ca-ATPASE (SERCA) is an ER transmembrane protein that pumps calcium back into the ER; without this pump, calcium recruitment in the ER cannot occur. Further regulation can occur within the ER: Calreticulin and calsequestrin are two major Ca²⁺-binding proteins inside the ER membranes that act as Ca²⁺ buffers. ^29–31 Both calreticulin and calsequestrin play a critical role in Ca²⁺ homeostasis in the lumen of the ER. ^32,33 Calsequestrin is a regulator of RyR activity and many studies show that it regulates protein synthesis ^34,35 while calreticulin is a versatile lectin-like chaperone and also has been implicated in a variety of cellular functions. ³³ The major function of these Ca²⁺-binding chaperones is to increase the Ca²⁺ storage capacity of the ER lumen. Therefore, expression of such proteins is vital for active calcium signaling. Scrutiny of these expression patterns in serum-maintained and serum-deprived cells will be a worthwhile topic of study in the future. 

Having demonstrated that the ER store can be modulated by serum deprivation it was important to determine a functional role for calcium signaling in pterygium. The agonists used to assess changes in calcium signaling can stimulate a number of pathways; therefore, explicitly linking calcium mobilization to a specific function by a ligand isn't straightforward. Further investigation of the signaling pathways activated by these ligands and their functional roles in pterygium will be a valuable topic of investigation in the future. However, to demonstrate a functional consequence of calcium signaling, it was necessary to utilize thapsigargin to deplete the store and disrupt signaling. Using this approach, we have shown that disruption of the ER store and thus calcium signaling could affect cell growth and migration of pterygial-derived fibroblast. This suggests that regulation of the ER store is an important consideration in the management of pterygia. These data tie in well with studies in other cells and tissues that have demonstrated that the ER store is required for cell division ³⁶ and migration. ^37,38 Persistent depletion of the store can result in reduced protein synthesis, ER stress, and apoptosis. ^36,38–40 It is feasible that serum starvation over prolonged periods of time could lead to an increasingly diminished store. This in turn could affect basic functions associated with the progression of pterygium. In the first instance, a reduction in migration and proliferation would be predicted followed by ER stress–lead processes such as reduced protein synthesis and potentially cell death by apoptosis. ^36,38–40 Applying this principle to the clinic, strategies that reduce the blood supply to the pterygium, such as anti-VEGF, ²⁶ could facilitate a reduction of calcium in the ER store; when this reaches a threshold, cell functions will be impaired leading to reduced growth and potentially cell death. Future work into calcium signaling and the store per se should examine the effects of long-term depletion of the ER store and ER stress–related events in relation to cell growth, migration and survival of pterygial fibroblasts in relation to putative therapies. 

Acknowledgments

The authors thank Julie Eldred for technical assistance, advice, and discussion. We also thank Sue Zabari for her administrative role in the organization of this project. 

Supported by the Humane Research Trust. 

Disclosure: C. Fang, None; C.D. Illingworth, None; L. Qian, None; I.M. Wormstone, None