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Published in final edited form as: Exp Eye Res. 2019 Aug 26;187:107776. doi: 10.1016/j.exer.2019.107776

Vitamin D receptor and metabolite effects on corneal epithelial cell gap junction proteins

Xiaowen Lu a, Zhong Chen a, Sarah Vick a, Mitchell A Watsky a,b,*
PMCID: PMC6759376  NIHMSID: NIHMS1538911  PMID: 31465769

Abstract

Vitamin D is a fat-soluble prohormone that can be activated both systemically and within individual tissues. Our lab has previously demonstrated that the corneal epithelium can activate vitamin D and that the vitamin D metabolites 1,25(OH)2D3 and 24R,25(OH)2D3 can affect corneal epithelial migration, proliferation, and tight and gap junction function. These vitamin D-derived metabolites signal through the vitamin D receptor (VDR). The purpose of this study was to specifically determine the effects of 1,25(OH)2D3 and 24R,25(OH)2D3 on corneal epithelial cell gap junction proteins. Connexin (Cx) 26, 30 and 43 protein expression was detected in a human corneal epithelial cell line (HCEC), wild type and vitamin D receptor knockout (VDR −/−) mouse corneas, and cultured mouse primary epithelial cells (MPCEC). In vitro gap junction function was assessed using the scrape loading/dye transfer assay. HCEC and MPCEC were treated with 1,25(OH)2D3 or 24R,25(OH)2D3. Western blotting was used to detect gap junction proteins. Vitamin D3 effects on epithelial intracellular Ca++ (Cai++) were determined using the dye Cal-520. Cx26 and Cx43 protein levels were significantly increased in HCEC and MPCEC treated with both 1,25(OH)2D3 and 24R,25(OH)2D3. Cx30 and Cx43 protein levels were also significantly increased in VDR −/− MPCEC. In vitro gap junction connectivity was significanlty enhanced in HCEC and MPCEC cultured with 24R,25(OH)2D3 and 1,25(OH)2D3. Cai++ was not affected by 1,25(OH)2D3 or 24R,25(OH)2D3 in HCEC or MPCEC. We conclude that both 1,25(OH)2D3 and 24R,25(OH)2D3 are positive regulators of connexin proteins and gap junction communication in the corneal epithelium. These vitamin D metabolites appear to signal through both VDR-dependent and -independent pathways. The effects of vitamin D on corneal epithelial gap junctions do not seem to be dependent on Cai++.

Keywords: Gap junction, Corneal epithelium, Connexins, Vitamin D

1. Introduction

Gap junctions are present in all corneal cell types and have been shown to have a critical role in cell phenotype determination (Watsky, 1995; Williams and Watsky, 1997, 2002). Connexin (Cx) proteins assemble into groups of six to form hemichannels or connexons, and two hemichannels then combine to form a gap junction (Yeager and Nicholson, 1996). Gap junction communication in the human corneal epithelium is mediated by Cx26, −30, −31.1, and −43 (Shurman et al., 2005; Williams and Watsky, 2002). Eight connexin transcripts (Cx26, −30.3, —31, —31.1, −33, −37, −43, −50) have been identified in the rat cornea (Laux-Fenton et al., 2003). Cx43 has been detected in rabbit cornea (Williams and Watsky, 2004), and it has been associated with inflammatory responses and wound healing in injured corneas (Moore et al., 2013). Cx43 and −50 have been detected in the mouse corneal epithelium (Matic et al., 1997).

Cx protein mutations often result in functional or developmental abnormalities, and have recently been associated with ocular disorders, including Cx43 mutations in oculodentodigital dysplasia, Cx26 mutations in keratitis-ichthyosis-deafness syndrome, and Cx46 and Cx50 mutations in congenital zonular pulverulent cataract (Caceres-Rios et al., 1996; Hansen et al., 2007; Kjaer et al., 2004). It has also been demonstrated that mRNA and protein levels of Cx31.1 and Cx43 are significantly upregulated in human cornea samples affected by chemical burns and infection (Zhai et al., 2014).

Vitamin D deficiency is extremely prevalent world-wide, and has been associated with numerous health problems including cardiovascular disease (Pekkanen et al., 2015), cancer (Ordonez Mena and Brenner, 2014), and immune/autoimmune disorders (Alsalem et al., 2014; Kosmowska-Miskow, 2014). Our lab has been examining the influence of vitamin D on corneal physiology and wound healing. Using the fluorescence recovery after photobleaching method, we examined the influence of vitamin D receptor knockout (VDR−/−) on epithelial gap junction communication in intact mouse corneas. We determined that the superficial epithelium was affected by VDR−/−, showing significantly reduced diffusion coefficients (Lu and Watsky, 2014). Our laboratory has also demonstrated that 1,25(OH)2D3 increases tight junction expression in human corneal endothelial cells (Elizondo et al., 2014).

In the vitamin D metabolic pathway, both 25(OH)D3 and 1,25(OH)2D3 can be acted on by 24-hydoxylase to form 24R,25(OH)2D3. 24R,25(OH)2D3 has historically been considered the final metabolic byproduct of vitamin D metabolism, with minimal physiological activity. Only recently has it been recognized that 24R,25(OH)2D3 is more than a metabolic byproduct of vitamin D metabolism (Lu et al., 2017), with reported physiological activity in bone (Curtis et al., 2014; van der Meijden et al., 2014). Human microarray data determined that 24R, 25(OH)2D3 modulates cell differentiation and proliferation, and influences the expression of several membrane proteins. Our lab determined that 24R,25(OH)2D3 is one of the most prevalent vitamin D metabolites in the rabbit eye (Lu et al., 2015). Our studies also determined that dietary vitamin D supplementation resulted in significantly increased 24R, 25(OH)2D3 levels in the eye (Lin et al., 2012). Recently, we also determined that 24,25(OH)2D3 increases corneal CYP24A1 and CYP27B1 protein expression, and stimulates proliferation and migration of mouse and human corneal epithelial cells (Lu et al., 2017).

The actions of vitamin D are primarily mediated through the stereospecific interaction of 1,25(OH)2D3 with VDR, a nuclear receptor and member of the steroid/thyroid receptor subfamily (Kubota et al., 2001). 1,25(OH)2D3 has been shown to affect the formation and degradation of gap junctions in an androgen responsive prostate cancer cell line (Kelsey et al., 2014). 1,25(OH)2D3 induces gap junctional intercellular communication in human skin fibroblasts, including a VDR-dependent increase in Cx43 protein and Cx43 mRNA levels (Clairmont et al., 1996). Previous results suggest that 1,25(OH)2D3 affects gap junctional intercellular communication at the level of transcription or stability of mRNA via the nuclear VDR (Clairmont et al., 1996).

The current study was designed to examine the effects of 1,25(OH)2D3 and 24R,25(OH)2D3 on corneal epithelial cell connexin proteins and gap junctions. Moreover, we determined if these effects are VDR-dependent.

2. Methods

2.1. Materials

1,25(OH)2D3 and 24R,25(OH)2D3 were purchased from Enzo Life Sciences (Farmingdale, NY) (catalog numbers BML-DM200-0050, BML-DM300-0050). Cx26 and Cx30 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) (catalog numbers sc130729, sc-84801). Cx43 and GAPDH antibodies were purchased from Cell Signaling Technology (Danvers, MA) (catalog numbers 3512, 5174). The Pdia3 (Protein disulfide isomerase family A member 3) antibody (Ab099) (Nemere et al., 2010) was a kind gift of Dr. Ilka Nemere. Prestained protein markers were obtained from Bio-Rad (Hercules, CA). Polyvinylidene difluoride (PVDF) membrane and the enhanced chemiluminescence (ECL) detection system were obtained from Bio-Rad (Hercules, CA).

2.2. Human corneal epithelial cell line

The immortalized human corneal epithelial cell line (HCEC) has been previously described (Griffith et al., 1999; Lu et al., 2017; Yin et al., 2011). HCEC were grown on standard culture plates until confluent (Griffith et al., 1999). All cells were grown in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) supplemented with FBS (3%), 1% Insulin-Transferrin-Selenium (ITS, Fisher Scientific, Waltham, MA), and 40 μg/mL gentamicin (Fisher Scientific, Waltham, MA). Cells were subpassaged using trypsin (Sigma, Ann Arbor, MI) digestion, seeded in 35 mm dishes (Fisher Scientific, Waltham, MA), and cultured in a humidified incubator at 37 °C with 5% CO2. Culture medium was replaced with fresh DMEM medium plus 3% serum every 2 days. While this cell line cannot be verified by STR profiling because the source cells are not available for genetic comparison, HCEC are routinely checked by PCR analysis for mRNA expression of the epithelial markers keratin 3 and keratin 12, and cell morphology was always examined and verified.

2.3. Human primary corneal epithelial cells (HPCEC) and mouse primary corneal epithelial cells (MPCEC)

Primary human epithelial cells were separatly obtained from two de-identified donor corneal rims (one female and one unidentifed sex) courtesy of Dr. Amy Estes from the Department of Ophthalmolgy, Medical College of Georgia, Augusta University and The Eye Guys, Eye Physicians and Surgeons of Augusta, GA. Wildtype (VDR+/+) and VDR−/− mice were obtained and bred from the Jackson Labs (Strain: B6.129S4-Vdrtm1Mbd, Stock No: 006133). Mixed sexes were used. All animal studies were approved by the University IACUC, and animals were treated according to the ARVO statement for the Use of Animals in Ophthalmic and Visual Research. Primary corneal epithelial cell cultures were established using a modification of the established explant culture method (Kobayashi et al., 2009; Ma et al., 2009). Briefly, the eyes were enucleated, and cornea buttons were obtained using a pathogen-free disposable punch (2.5 mm) and washed with Ca++ free PBS (pH 7.2). Each cornea was cut in half and placed in a 35 mm dish (Fisher Scientific, Waltham, MA) with the epithelial side up. After approximately 5 min in a laminar air-flow culture hood, the half corneal button securely attached to the bottom of the plate, and 1.5 mL of DMEM (Life technology) with 10% serum containing 40 μg/mL gentamicin (Life technologies, NY), 1% ITS (Fisher Scientific, PA), and 100ng/mL cholera toxin (LIST Biological Laboratories, Inc., Campbell, CA) was added and the tissue was cultured in a humidified incubator at 37 °C with 5% CO2. Culture medium was replaced every 2 days. After 7–10 days cells were nearly 100% confluent.

Cells were passaged using 0.25% trypsin (Fisher Scientific, Waltham, MA), centrifuging at 500 g for 5 min, and subculturing in DMEM with 3% serum containing 40 μg/mL gentamicin (Life technologies, NY), 1% ITS (Fisher Scientific, Waltham, MA), and 100ng/mL cholera toxin (LIST Biological Laboratories, Inc., Campbell, CA). Primary cell passages 3–6 were used for these experiments. All plates were inspected prior to use under the microscope to ensure minimal contamination by stromal cells.

2.4. Dye transfer assay

Gap junction connectivity was assessed using the scrape loading/ dye transfer assay. HCEC, VDR+/+ and VDR−/− MPCEC were grown to 100% confluence in 35-mm dishes. After medium was removed, 5 (6)-carboxyfluorescein (CF) dissolved in PBS (0.5 mg/mL) was added to the dish, and scrape wounds were immediately created using a surgical blade. Five minutes later, the cells were washed with PBS to remove excess dye and were fixed with 3.7% formalin. An Olympus fluorescence microscope (model IX73) was used to view CF-positive cells. Dye spread was defined as the distance of CF-positive cells from the scrape line as measured using the Olympus CellSens Dimension 1.11 software package. The reported dye spread ratio equals the measured dye spread of treated/control cells.

2.5. Immunofluorescence studies

Eyes were enucleated from euthanized mice and prepared for cryosectioning. Eyes were flash frozen in liquid nitrogen and embedded in optimal cutting temperature compound (Tissue-Tek, Sakura Finetek, Torrance, CA, USA). 10 μm-thick cryosections were fixed for 10 min in 4% paraformaldehyde (4% PFA) and blocked with 10% goat serum in 0.1% Triton X-100/PBS for 1 h at room temperature. Cryosections were incubated with primary antibodies (anti-Cx26, anti-Cx30 and anti-Cx43 followed by incubation with secondary antibodies. Cryosections were examined using a Zeiss LSM 780 upright laser-scanning confocal microscope equipped with ZEN lite software.

2.6. Protein extraction and western blot analysis

Protein was isolated from confluent HCEC and VDR+/+ and VDR−/− MPCEC cells grown on 35 mm dishes and from freshly isolated epithelial cells scraped from mouse corneas using a Gill corneal knife (Storz Ophthalmic Instruments, Rhode Island, New England). After washing cells with PBS, they were exposed to lysis buffer (50 mM Tris-HCl [pH 8], 150 mM NaCl, 0.02% N3Na, 100μg/mL phenylmethylsulfonyl fluoride, 1% NP-40, 50 mM NaF, 2 mM EDTA, and protease inhibitor [Sigma, Ann Arbor, MI]). Cell lysates were collected and Western blotting was performed as previously described (Yin et al., 2011). Blots were labeled with Cx26, Cx30, Cx43 or Pdia3 antibodies at a dilution of 1:1000. Membranes were washed and then incubated with anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:3000). Detection was performed using the enhanced chemiluminescence method (Pierce Biotechnology, Rockford, IL). To ensure equal protein loading, nitrocellulose membranes were labeled with GAPDH antibody at a dilution of 1:6000 (Cell Signaling Technology, Danvers, MA).

2.7. Vitamin D3 effects on Intracellular Calcium concentration

Human corneal rims (n = 2 female, n = 2 male), HCEC, and human and mouse primary corneal epithelial cells were loaded with Cal-520 dye (AAT Bioquest, Sunnyvale, California) and visualized under a Zeiss 780 inverted confocal microscope (Ex/Em = 490/525 nm). Cells were treated with 1,25(OH)2D3 (20 nM) or 24R, 25(OH)2D3 (100 nM), followed by ATP (5 μM) as a positive control. Baseline images of epithelial cells were recorded for 3 min at 1 image/10 s, followed by up to 20 s of 1 image/500 msec immediately before and after vitamin D exposure. To view longer term vitamin D effects, continued imaging was done for 60 min at 1 image/10 s, and positive control images were then collected as 1 image/500 msec for 5 min before and after ATP exposure followed by 5 min of 1 image/10 s. Dye intensity was measured over the entire time frame. Vitamin D trials were only included in the final analysis if there was a positive ATP response.

2.8. Mycoplasma testing

Cultures were found to be mycoplasma negative using the MycoProbe ™ Mycoplasma Detection Kit (R&D Systems Inc, Minneapolis, MN. Catalog Number CUL001B).

2.9. Statistical analysis

All data are provided as the x¯±SE of at least three experiments. Where applicable, differences between two groups were compared using the unpaired Student's t-test. P < 0.05 was considered statistically significant.

3. Results

3.1. Gap junction connectivity

Our previous study demonstrated that VDR knockout resulted in reduced superficial corneal epithelial cell gap junction dye diffusion rates (Lu and Watsky, 2014). To examine the direct influence of vitamin D metabolites on HCEC and VDR−/− MPCEC, changes in gap junction connectivity was determined by dye transfer following treatment with 24R,25(OH)2D3 or 1,25(OH)2D3 for 18 h as measured by the dye spread ratio (Fig. 1). 24R, 25(OH)2D3 treatment resulted in increased dye migration (μm2; mean ratio ± SEM) from the scrape line in HCEC (2.76 ± 0.24), VDR+/+ (1.22 ± 0.09), and VDR−/− MPCEC (1.47 ± 0.11) compared to untreated controls (T-test, P < 0.05). 1,25(OH)2D3 increased the dye migration ratio in VDR+/+ (1.28 ± 0.22) and VDR−/− MPCEC (1.28 ± 0.09) (P < 0.05). 1,25(OH)2D3 had no effect on HCEC dye migration.

Fig. 1. Scrape loading/dye transfer assay in HCEC, VDR+/+, and VDR−/− MPCEC.

Fig. 1.

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Representative fluorescence and bright-field images of control, 10 nM 1,25(OH)2D3 and 50 nM 24R,25(OH)2D3 treated (A) HCEC, (B) VDR+/+, and (C) VDR−/− MPCEC cells (n = 5). (D) Normalized scrape wound dye spread ratio results for human and VDR+/+ and VDR−/− mouse epithelial cells. 24R,25(OH)2D3 significantly increased gap junction communication in all cell types, while 1,25(OH)2D3 only increased communication in MPCEC (t-test, x¯±SE, *P < 0.05, **P < 0.01 indicates statistical significance as compared to control, n = 3).

3.2. VDR knockout effects on connexin protein expression

Immunohistochemical localization and western blotting were used to determine if VDR knockout directly affects gap junction protein expression. Immunohistochemical localization of the connexin proteins demonstrates that Cx26, −30 and −43 are primarily expressed in the basal and intermediate layers (Fig. 2A–C). The intensity of Cx26, −30 and −43 immunostaining was decreased in VDR−/− mice. Cx43, Cx30, and Cx26 relative protein expression levels (0.41 ± 0.24, 0.22 ± 0.17, 0.51 ± 0.15, respectively; x¯±SE) were all significantly decreased in VDR−/− mice as compared to VDR+/+ controls (P < 0.05, Fig. 2D and E).

Fig. 2.

Fig. 2.

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Representative immunostaining and western blots demonstrating the effect VDR−/− on connexin localization and protein expression. We observed (A) Cx26, (B) Cx30, and (C) Cx43 immunostaining in mouse corneal epithelium that were all reduced in VDR−/− mice. DAPI nuclear staining is blue and negative controls have no primay antibody. (D,E) Representative western blots and density plot from mouse cornea tissue (n = 3 VDR+/+, n = 3 VDR−/− from mixed sexes) also demonstrating reduced Cx26, Cx30, and Cx43 expression in cells from VDR−/− mice (t-test, x¯±SE, *P < 0.05, n = 3). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

3.3. Vitamin D3 effects on connexin expression in VDR+/+ MPCEC

VDR+/+ MPCEC were treated with 1,25(OH)2D3 and 24R,25(OH)2D3 to determine the effects of vitamin D metabolites effects on gap junctional proteins in MPCEC with intact VDR. 1,25(OH)2D3 and 24R,25(OH)2D3, respectivel (x¯±SE), significantly increased Cx26 (1.32 ± 0.27, 1.34 ± 0.27) and Cx43 (1.41 ± 0.26, 1.29 ± 0.31) protein expression levels in VDR+/+ MPCEC relative to untreated cells (P < 0.01). There was no significant difference in the Cx30 protein expression level between vitamin D-treated versus control cells (Fig. 3).

Fig. 3. Western blot analysis of Cx protein expression in VDR+/+ MPCEC treated with vitamin D3.

Fig. 3.

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(A) Cx26 and (C) Cx43 protein expression was significantly increased in VDR+/+ MPCEC cultured with 1,25(OH)2D3 or 24R,25(OH)2D3. (B) There were no significant differences in Cx30 expression. *P < 0.05 indicates statistical significance as compared to control (n = 3).

3.4. Vitamin D3 effects on connexin expression in VDR−/− MPCEC

Our previous work demonstrated VDR-independent effects of vitamin D metabolites on corneal epitheial cells (Lu et al., 2017). VDR−/− MPCEC were treated with 1,25(OH)2D3 and 24R,25(OH)2D3 to determine the effects of vitamin D metabolites effects on gap junctional proteins in MPCEC without intact VDR. 1,25(OH)2D3 and 24R,25(OH)2D3, respectively (x¯±SE), significantly increased Cx30 (1.53 ± 0.15, 1.28 ± 0.23) and Cx43 (1.45 ± 0.30, 1.28 ± 0.10) levels in VDR−/− MPCEC relative to untreated cells (P < 0.01). These was no significant difference in Cx26 protein expression between vitamin D-treated versus control cells (Fig. 4).

Fig. 4. Western blot analysis of Cx protein expression in VDR−/− MPCEC treated with vitamin D3.

Fig. 4.

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(A) There was no significant difference in Cx26 protein expression in vitamn D3-treated cells. (B) Cx30 and (C) Cx43 protein expression was significantly increased in VDR−/− MPCEC cultured with 1,25(OH)2D3 or 24R,25(OH)2D3. *p < 0.05 indicates statistical significance as compared to control (n = 3).

3.5. Vitamin D3 effects on connexin expression in HCEC

Western blotting was used to determine if vitamin D affects human corneal epithelial gap junction protein expression. 1,25(OH)2D3 and 24R,25(OH)2D3, respectively (x¯±SE), significantly increased HCEC Cx26 (1.42 ± 0.24, 1.48 ± 0.17) and Cx43 (1.93 ± 0.45, 2.52 ± 0.48) protein levels relative to untreated cells (P < 0.05, Fig. 5). However, Cx30 protein expression (0.70 ± 0.10) was significantly decreased in HCEC cultured with 1,25(OH)2D3 cells (P < 0.05), with no difference in HCEC cultured with 24R, 25(OH)2D3.

Fig. 5. Western blot analysis of Cx protein expression in HCEC treated with vitamin D3.

Fig. 5.

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(A) Cx26 and (C) Cx43 protein expression was significanlty increased in HCEC cultured with 1,25(OH)2D3 and 24R,25(OH)2D3. (B) Cx30 protein expression was significantly decreased in HCEC cultured with 1,25(OH)2D3 and unchanged in 24R,25(OH)2D3-treated cells. *P < 0.05 indicates statistical significance as compared to control (n = 3).

3.6. Pdia3 expression in HCEC, HPCEC, and MPCEC

Because the Pdia3 protein has been shown to be an alternative vitamin D receptor in some cells, western blotting was used to determine if Pdia3 is present in corneal epithelial cells. Pdia3 was detected in HCEC, HPCEC, and MPCEC (Fig. 6).

Fig. 6. Western blot detection of Pdia3.

Fig. 6.

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Pdia3 was detected in HCEC, HPCEC, and MPCEC by Western blot.

3.7. Vitamin D3 effects on Cai++

Vitamin D is well known for its calcemic effects in the body. Calcium imaging was performed in intact corneal rim epithelial cells and cultured epithelial cells to determine if vitamin D metabolites have accute effects on corneal epithelial Cai++. Fig. 7 shows representative fluorescence images and dye intensity plots from an intact human corneal rim epithelia cut in half and exposed to 1,25(OH)2D3 or 24R,25(OH)2D3, followed by ATP as a positive control. Cai++ was not affected by addition of 1,25(OH)2D3 or 24R,25(OH)2D3 to the culture medium, while the positive control ATP caused a large increase in dye intensity, indicating a Cai++ increase. Similar results were observed in HCEC and primary human corneal epithelial cells (not shown). Thus, it is unlikely that changes in intracellular Ca++ levels are linked to the gap junction results.

Fig. 7. No effect of vitamin 1,25(OH)2D3 and 24R,25(OH)2D3 on intracellular Ca++ levels in human corneal rim epithelial cells.

Fig. 7.

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Representative fluorescence intensity profiles of Cal520-loaded intact human corneal rim epithelia are shown in A and B. Cells were stained with Cal-520 dye and exposed to (A) 1,25(OH)2D3 (20 nM) or (B) 24R,25(OH)2D3 (100 nM) while Cal-520 fluorscence intensity was being measured. 5 μM ATP was used a positive control. Vitamin D did not affect intracellular Ca ++ concentrations.

4. Discussion

4.1. Cx26, −30 and −43 in VDR−/− mouse corneal epithelium

Cx26 and Cx30 proteins co-localize in most gap junction plaques and often form heteromeric channels (Ahmad et al., 2003). In the current study, Cx26, −30 and −43 were detected in mouse and human corneal epithelium. This is the first report of Cx26 in the mouse corneal epithelium. A Cx26 mutation has been identified as the cause of keratitis-ichthyosis-deafness syndrome in humans, which is characterized by keratitis and chronic progressive corneal neovascularization, skin hyperplasia and increased carcinogenic potential (Schutz et al., 2011).

Cx26, −30 and −43 were decreased in VDR−/− mouse corneal epithelium. The reduced Cx protein levels in VDR−/− mice helps to explain the low gap junction diffusion coefficient in VDR−/− superficial epithelial cells seen in our previous study (Lu and Watsky, 2014). In addition, this reduced connexin expression may be associated with the attenuated corneal epithelial wound healing we have demonstrated in VDR−/− mice (Elizondo et al., 2014). Cx 43 has been found to contribute to the corneal cell growth and differentiation (Dong et al., 1994; Wolosin et al., 2004). Moreover, Cx26 and Cx43 were found to be increased in rabbit corneas after laser photorefractive keratectomy (Ratkay-Traub et al., 2001), and Cx26, Cx31.1, and Cx43 were increased in chemically burned and infected human corneas (Zhai et al., 2014). It is postulated that the connexin increases in these circumstances are associated with the wound healing response.

4.2. Effects of 1,25(OH)2D3 and the VDR on corneal epithelial gap junctions

1,25(OH)2D3 increased gap junction-related dye diffusion and connexin protein levels in MPCEC and HCEC, indicating that 1,25(OH)2D3 plays a role in cell communication in the cornea. 1,25(OH)2D3 and 24,25(OH)2D3 significantly increased gap junction dye diffusion in each of the models tested except for HCEC. We have previously noted species differences in the effects of these metabolites when comparing human and mouse cells (Lu et al., 2017) and this is likely the interpretation of these results. 24,25(OH)2D3 increased the dye diffusion much more in HCEC compared to in mouse cells, further supporting this interpretation. Interestingly, the HCEC 24,25(OH)2D3 response was several fold higher than any response initiated by1,25(OH)2D3. This confirms work by our lab and others demonstrating that 24,25(OH)2D3 is a highly active vitamin D metabolite (see below).

Table 1 summarizes the effects of 1,25(OH)2D3 and 24,25(OH)2D3 on connexin proteins in each of the models used in this study, including MPCEC from VDR−/− mice. The use of VDR−/− mice and cultured cells obtained from these mice is a common model used to study vitamin D deficiency because of the specificity of the VDR for 1,25-dihydroxyvitamin D. Significant effects of 1,25(OH)2D3 on VDR−/− MPCEC indicates a likely alternative vitamin D signaling pathway. The current study and our previous work indicate that both VDR-dependent and VDR-independent signaling pathways are present in corneal epithelial cells. As described earlier, our previous studies in VDR−/− mice demonstrated attenuated corneal epithelial wound healing (Elizondo et al., 2014) and a reduced superficial squamous corneal epithelial cell gap junction diffusion coefficient compared to VDR+/+ mice (Lu and Watsky, 2014). These appear to be VDR-dependent effects. On the other hand, 1,25(OH)2D3 significantly increased cultured human epithelial cell proliferation in VDR−/− mice, and had significant effects on vitamin D metabolizing enzymes in VDR−/− mice (Lu et al., 2017), indicating VDR-independent effects. In the current study, Cx26, Cx30 and Cx43 protein expression levels were all found to be significantly decreased in VDR−/− corneas compared to wild type controls, demonstrating VDR-dependent signaling. Alternatively, significant changes in Cx protein levels and dye spread in vitamin D-treated VDR−/− MPCEC indicates VDR-independent signaling. As summarized in Table 1, Cx26 and −43 were both elevated by 1,25(OH)2D3, while Cx30 was decreased by 1,25(OH)2D3 in HCEC, and increased by 1,25(OH)2D3 in VDR−/− MPCEC. The vitamin D3-stimulated increase in Cx30 levels in VDR−/− MPCEC indicate not only that an alternative vitamin D signaling pathway is present in these cells, but also that VDR is likely a negative regulator of Cx26. These studies indicate that the corneal epithelial gap junction protein levels can be mediated by vitamin D through VDR or through a secondary vitamin D receptor. Such a secondary vitamin D receptor, Pdia3, has been identified in other tissues (Sequeira et al., 2012; Wang et al., 2010, 2014), and in this study we demonstrate that Pdia3 is present in corneal epithelial cells.

Table 1.

Summary of 1, 25(OH)2D3 and 24R,25(OH)2D3 effects on Connexins.

Connexins Cell &VitD3
HCEC VDR+/+ MPCEC VDR−/− MPCEC
1,25(OH)2D3 24R,25(OH)2D3 1,25(OH)2D3 24R,25(OH)2D3 1,25(OH)2D3 24R,25(OH)2D3
Cx26 a a a a NC NC
Cx30 – NC NC NC a a
Cx43 a a a a a a
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a

Significantly increased, - significantly decreased, NC no change.

4.3. 24,25(OH)2D3 is a biologically active vitamin D metabolite in the cornea

It is generally believed that 1,25(OH)2D3 is the most biologically active vitamin D metabolite, and classical nuclear vitamin D receptor is considered the primary receptor for 1,25(OH)2D3. 24,25(OH)2D3 was considered to be an inactive form of vitamin D3, with few previous studies examining the possible function(s) of 24R, 25(OH)2D3 in the cornea. In the few studies in any tissue type demonstrating a physiological role for 24R,25(OH)2D3, it was shown to be important during human mesenchymal stem cell maturation, and has an essential role in Ca++ mineralization gene expression (Curtis et al., 2014). Previous work from our laboratory demonstrated that 24R, 25(OH)2D3 stimulates both HCEC cell proliferation and migration, which are crucial for corneal epithelial wound healing. In the current study, 24R,25(OH)2D3 was found to increase gap junction communication in HCEC and VDR+/+ and VDR−/− MPCEC using the scrape loading/dye transfer assay. In addition, 24R,25(OH)2D3 increased Cx26 and Cx43 protein expression in these same cells. 24R,25(OH)2D3 also increased Cx30 and Cx43 in VDR−/− MPCEC, indicating that it too can signal through a VDR-independent pathway.

4.4. Vitamin D and corneal epithelia Ca++ transport

Vitamin D is well known for its calcemic effects in the body through its interaction with the VDR. For example, in the gut, vitamin D stimulates Ca++ transport (Wongdee and Charoenphandhu, 2015). The attenuation of gap junction function and connexin protein levels in VDR−/− mice and cells may be due to changes in intracellular Ca++ homeostasis. To address this possibility, we conducted experiments visualizing intracellular Ca++ using the Ca++-sensitive dye Cal-520 (AAT Bioquest) in human corneal rim epithelia, primary human epithelial cells and the human epithelial cell line. Intracellular Ca++ was not affected by addition of 1,25(OH)2D3 or 24R,25(OH)2D3 to any of the cells examined, thus Ca++ levels likely have no effect on the cell culture connexin/gap junction results.

In summary, both 1,25(OH)2D3 and 24R,25(OH)2D3 are positive regulators of connexin proteins and gap junction communication in the corneal epithelium. These vitamin D metabolites appear to signal through both VDR-dependent and -independent pathways. The effects of vitamin D on corneal epithelial gap junctions do not seem to be dependent on intracellular Ca++ levels.

Acknowledgements

We are grateful to Dr. Amy Estes (Department of Ophthalmology, Medical College of Georgia at Augusta University, Augusta, GA) and The Eye Guys, Eye Physicians and Surgeons of Augusta, GA for providing donor corneal rims. This work was supported by NIH Grant EY021747-06.

Footnotes

Disclosure

All authors have approved this final article as being true in representation of the experimental findings.

References

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