University of Birmingham Characterization of vitamin D production by human ocular barrier cells

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V itamin D 3 is a secosteroid synthesized from the conversion of the precursor 7-dehydrocholesterol (7DHC) in the skin to vitamin D 3 by ultraviolet (UV) B sunlight.To a lesser extent, vitamin D 3 can be obtained from the diet.Sufficient levels of vitamin D 3 are required for calcium absorption and maintenance of healthy bones. 1 In the circulation, vitamin D 3 is activated by two successive hydroxylation steps.The first occurs in the liver by a cytochrome P (CYP) 450 enzyme, 25hydroxylase (mitochondrial CYP27A1 and microsomal CYP2R1) to produce the major circulating form 25-hydroxyvitamin D 3 (25[OH]D 3 ).The second hydroxylation step occurs in the kidney, where 25-hydroxyviatmin D 1a-hydroxylase (CYP27B1) catalyzes the conversion of 25(OH)D 3 to its active form 1,25-dihydroxyvitamin D 3 (1,25[OH] 2 D 3 ).As a feedback mechanism, 24-hydroxylase (CYP24A1), which is induced by 1,25(OH) 2 D 3 , inactivates both forms of vitamin D 3 .The 1,25(OH) 2 D 3 action is mediated via the vitamin D receptor (VDR) that heterodimerizes with retinoid X receptor (RXR), a complex that regulates gene transcription through binding to vitamin D response elements (VDRE) in the promoter region of target genes. 2xtrarenal synthesis of vitamin D 3 has been reported in many tissues and cells, including barrier sites.4][5][6][7][8][9] In the circulation, 25(OH)D 3 is bound to vitamin D binding protein (DBP), which facilitates its cellular uptake by multi-ligand endocytic receptors megalin/cubilin expressed on the apical surface of polarized epithelial cells in many tissues. 102][13][14] Of note, VDR is critical for barrier formation in human skin and the integrity of mucosal barrier in mouse intestine. 15,16Recently, vitamin D 3 has been recognized as an immunomodulatory hormone that regulates innate and acquired immune responses. 17One regulatory role of 1,25(OH) 2 D 3 in the immune system (in vitro) involves the inhibition of proinflammatory cytokine production from CD4 þ T cells and the induction of a regulatory T cell phenotype. 18ndogenous conversion of 25(OH)D 3 has been shown to inhibit dendritic cell antigen presentation and chemotaxis. 19he ability to induce the production of host defense peptides (HDPs) in blood monocytes also supports an interaction between 1,25(OH) 2 D 3 and the immune system.The HDP LL-37 (cathelicidin) and human beta defensins (hBD1-4) can kill a wide range of Gram-negative and Gram-positive bacteria, as well as viruses.Moreover, HDPs are immunomodulatory molecules. 20They can act as chemotactic agents for T and dendritic cells, as well as their ability to induce T-helper 1 and 2 cytokines. 21cular immune privilege is maintained by a cluster of anatomic and physiological processes that protect the eye from sight-threatening infections and inflammatory responses. 22The sclera is the white, opaque, fibrous support to the global structure of the eye composed of a rich extracellular matrix (ECM) of collagen and elastic fibers, together with fibroblasts that are responsible for tissue remodeling and the regeneration of the ECM. 23On the posterior surface of the cornea, endothelial cells contribute to barrier function by mediating sodium transport, which maintains the cornea in a relatively dehydrated state, thereby preserving corneal transparency. 24he eye consists of two key vascular barriers that afford protection: the blood-aqueous barrier and the blood-retinal barrier.The blood-aqueous barrier consists of tight junctions between vascular endothelial cells of iridociliary body blood vessels, the nonpigmented ciliary epithelial cells complexed apically with the pigmented ciliary epithelium, and the posterior iris epithelium, whereas the blood-retinal barrier is composed of tight junctions of endothelial cells in retinal vasculature and those between the retinal pigment epithelial cells. 25o date, very few studies have examined the presence of vitamin D 3 synthesizing and metabolizing pathways in the eye, and whether these have a role in ocular barrier cell function by the autocrine production of vitamin D 3 .This study shows the expression and functionality of vitamin D 3 system in several human ocular barriers, intimating that vitamin D 3 may be important to the ocular barrier function and ocular immune privilege.

Cell Culture
All cells were grown in a humidified chamber at 378C and 5% CO 2 , and passaged by trypsinization.Penicillin (100 units/mL) and streptomycin (100 lg/mL) solution was added to all growth media (PAA Laboratories, Yeovil, UK).All cells were grown in 10% heat inactivated fetal calf serum (HIFCS) except human corneal endothelial cells, which were grown in 5% HIFCS (Biosera Ltd., Ringmer, UK).
Primary Human Scleral Fibroblasts (HSF).Primary human scleral fibroblasts were generated from redundant donated corneal scleral tissue following corneal transplant surgery.Acquisition of tissue was after approval from the local research ethics committee and adhered to the Declaration of Helsinki.The sclera was cut into 3-to 5-mm sections and layered in a T-25 cm 2 tissue culture flask (Sarstedt, Leicester, UK) for 3 to 5 days in fibro-medium (RPMI 1640 containing 1% MEM nonessential amino acid and 1% sodium pyruvate) until the fibroblasts grew out from the explant and maintained in fibro-medium (all Sigma-Aldrich, Dorset, UK).
Nonpigmented Ciliary Body Epithelial (ODM-2).The ODM-2 cell line was a kind gift from Dr Coca Prados (Department of Ophthalmology and Visual Sciences, Yale University, New Haven, CT, USA).Cells were cultured in high glucose (4 g/L) DMEM (PAA Laboratories) as described previously. 26

Reverse Transcription and PCR
Total RNA was isolated using RNeasy Mini Kit (Qiagen, Crawley, UK) according to manufacturer's instructions.The RNA concentration and purity were determined using Nanodrop spectrophotometer (Thermo Fisher Scientific, Loughborough, UK).Total RNA (1 lg) was reverse transcribed to cDNA using Taqman reverse transcription kit (Applied Biosystems, Warrington, UK) following manufacturer's instructions.The PCR was performed using GoTaq Flexi DNA polymerase system (Promega, Southampton, UK) in a total volume of 20 lL containing cDNA (1 lL for 18S and 2 lL for all other primers), 2 mM MgCl 2 , 31 GoTaq green Flexi Buffer, 0.5 U Gotaq DNA polymerase, 0.25 mM dNTP mix (Promega), 2.5 lM 18S, and 5 lM other forward and reverse primers (Alta Bioscience, Birmingham, UK).The PCR was performed using Gene Amp, PCR System 2700 (Applied Biosystems) as follows: 5 minutes at 948C, followed by three temperature cycles of 1 minute at 948C, annealing for 60 seconds, and 1 minute at 728C.For cubilin and megalin, preamplification was carried out at 958C for 5 minutes followed by cycles of denaturation at 948C for 45 seconds, annealing for 45 seconds, and extension at 728C for 45 seconds.All PCR reactions ended with a final extension step at 728C for 7 minutes.Primer sequences, taken from previous publications and cycling condition were as shown in Table 1.

Statistical Analysis
Statistical analyses were performed using GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA, USA).The Kruskal-Wallis test was used, followed by Dunn's multiple comparison test to compare differences between groups.Data are expressed as mean 6 SD and a P value < 0.05 was considered as statistically significant.

RESULTS
Ocular Barrier Cells Constitutively Express mRNA for Vitamin D 3 Metabolism Extrarenal expression of vitamin D 3 synthesizing and metabolizing components has been described in many organs.To examine the presence of these elements in ocular barriers, mRNA expression was examined by RT-PCR.The VDR was expressed strongly in all ocular cells tested (Fig. 1A).The CYP27B1 was expressed markedly by HCEC-12, ODM-2, and ARPE-19 compared to a weak expression in HSF.In contrast, CYP24A1 was expressed strongly by HSF and weakly by all other cell types.The CYP27A1 was expressed weakly in HCEC-12, ARPE-19, and ODM-2, but was undetected in HSF.The HSF, HCEC-12, and ARPE-19 highly expressed CYP2R1, whereas ODM-2 showed a weak expression.The mRNA expression of cubilin and megalin, the receptors required for the internalization of 25(OH)D 3 through DBP, also was investigated.Cubilin was expressed strongly in all cells, while megalin was only weakly expressed in HSF (Fig. 1B).To summarize, ocular barrier cells constitutively express mRNA for vitamin D 3 metabolism.

Ocular Barrier Cells Express Proteins for Vitamin D 3 Metabolism
Immunofluorescent staining of all cell types for the different vitamin D 3 elements was performed (Fig. 2, Supplementary Figs.S1A-D).The intensity of staining varied between cell types, but all cells were positive for all vitamin D 3 proteins.The VDR showed a diffuse mild cytoplasmic and abundant speckled nuclear staining.The expression was the strongest in ODM-2 and HCEC-12, and weaker in ARPE-19 and HSF, respectively.The CYP24A1 also was present in the cytoplasm, where the signal was strong in HCEC-12 and ODM-2, and weak in HSF and ARPE-19.The CYP27B1 showed an intense cytoplasmic pattern in ODM-2 and ARPE-19, while HSF showed moderate staining.Interestingly, in HCEC-12 cells, diffuse cytoplasmic staining was present, but most of the enzyme appeared within a perinuclear vesicle.

CYP27B1 Colocalizes With MPRs and TGN38 in HCEC-12 Cells
The YP27B1 normally is located in the inner membrane of the mitochondria. 3All the ocular cells demonstrated a staining pattern consistent with this expression, that is, cytoplasmic.
The exception was HCEC-12 cells, which not only expressed CYP27B1 in the cytoplasm, but the majority of the enzyme appeared as a vesicle adjacent to the nucleus.To further identify the nature of this vesicle-like structure, HCEC-12 were stained for Mannose 6 phosphate (M6P) receptors that are known to have a role in the transport of enzymes from the trans-Golgi network (TGN) to lysosomes 27 and typically shows perinuclear localization.In addition, TGN38 protein, part of the trans-Golgi network that directs proteins to secretory vesicles, lysosomes, or plasma membrane, also was exam- ined. 28The CYP27B1 colocalized to M6P receptors and TGN38 in HCEC-12 (Fig. 3A).The cytoplasmic staining, together with the perinuclear punctated presence (Fig. 3B) confirm CYP27B1 presence in the TGN in HCEC-12 cells.
Ocular Barrier Cells Can Convert Inactive Vitamin D 3 to the Active Form 1,25(OH) 2 D 3 Ocular barrier cells have the molecular components necessary to produce 1,25(OH) 2 D 3 locally.We next examined the functional capacity of CYP27B1 and its ability to convert inactive 25(OH)D 3 (10 À7 M) into active 1,25(OH) 2 D 3. The rate of conversion was variable among the different cells, where the highest levels were produced by HCEC-12 followed by HKC8 then ODM2, while the lowest levels were seen in ARPE-19 and none was detected in HSF (Fig. 4).
Substrate conversion in each cell type was inhibited by pretreatment with 10 À5 M ketoconazole and the inhibition is statistically significant in cells that produced more than 200 pmol/L/mg protein of 1,25(OH) 2 D 3 (HCEC-12, and ODM-2).

DISCUSSION
This study showed that human ocular barrier epithelial cells and scleral fibroblasts constitutively express the receptor and the metabolic enzymes required in the vitamin D 3 pathway, and for the first time to our knowledge that all these cell types can endogenously produce 1,25(OH) 2 D 3 when treated with physiological concentrations of its precursor 25(OH)D 3 (Table 2).These findings supported a recent report describing CYP27B1 and VDR mRNA in primary human corneal epithelium (PHCEC), while treatment with 25(OH)D 3 or 1,25(OH) 2 D 3 enhanced barrier function as shown by decreased inulin permeability and increased transepithelial resistance in vitro. 29In a separate study, a human corneal limbal epithelial cell line treated with UVB and 7DHC showed an increase in 1,25(OH) 2 D 3 production. 30Previous studies also have shown that corneal epithelial cells and RPE-choroid express the molecular elements for vitamin D 3 , but there were no data on 25(OH)D 3 conversion. 31s stated, vitamin D 3 synthesizing components (VDR, CYP27B1, and CYP24A1) are expressed widely in many human epithelial cells.In agreement with the speckled cytoplasmic and nuclear VDR staining in ocular barrier cells and sclera fibroblasts, VDR has been described in cytoplasm and nucleus of bladder epithelium as well as skin fibroblasts. 11,32The cytoplasmic expression of CYP27B1 in most of the ocular barrier cells is in accordance with similar expression in bladder epithelium.Conversely, the punctate perinuclear staining of CYP27B1 in HCEC-12 can be compared to the cytoplasmic localization of internalized Alexa-DBP in the perinuclear space of human breast cancer epithelial cells T-47D, where it colocalized with lysosomes, which suggested that DBP traffics through endosomes and lysosomes. 33The expression of CYP27B1 in TGN could be part of this enzyme's trafficking in these cells.Membrane-bound CYP450 proteins can recycle through Golgi before reaching their destined locations. 34lthough ocular barrier cells show constitutive expression of mRNA and protein for CYP24A1, this mitochondrial enzyme is not constitutively expressed in all peripheral organs and tissues.Some cells, such as skin keratinocytes, primary respiratory epithelial cells, and colonic epithelial cell line, only express CYP24A1 in response to 25(OH)D 3 or 1,25(OH) 2 D 3 .Keratinocytes demonstrate a strong up-regulation following exposure to UVB light in the presence of 7DHC. 4,13,35The CYP24A1 also is induced after treatment with 25(OH)D 3 sufficient serum in macrophages. 20ur study showed that ocular barrier epithelial cells can convert inactive vitamin D 3 to its active form.With the exception of ARPE-19, ocular barrier cells, are able to produce significant levels of active 1,25(OH) 2 D 3 (>0.4 3 10 À9 M) at 24 hours.This rate of conversion is comparable to that of primary   Ocular barrier cell lines are shown with mRNA expression, immunofluorescence staining intensity, and rate of 25D 3 conversion with the highest as þþþ and the lowest as þ.The HKC-8 cell line was used as a positive control for the expression of vitamin D 3 pathway components and 25(OH)D 3 conversion.Thyroid mRNA was used a positive control for the expression of cubilin and megalin.ND, not done.respiratory epithelial cells, bladder, and mammary epithelial cell lines. 4,11,36These rates are much higher than those produced by human kidney cortical collecting duct (HCD) cell line. 37As depicted in Figure 5 Ocular barrier epithelial cells appear to be efficient in producing 1,25(OH) 2 D 3 provided they have the substrate available.Despite the requirement of 25(OH)D 3 for CYP27B1 to convert it to 1,25(OH) 2 D 3 , ocular barrier cells potentially can initiate the vitamin D 3 pathway as early as the first 25hydroxylation step via CYP27A1 and CY2R1.Extrahepatic expression of CYP27A1 and CY2R1 also has been reported previously in other tissues. 39,40With its broad substrate specificity, CYP27A1 also is involved in cholesterol metabolism.The CYP27A1 is expressed in the retinal pigment epithelium and protects the retina from the toxic 7-ketocholesterol, a metabolite of 7-DHC as a result of photooxidation.Although CYP27A1 was expressed weakly by ocular barrier cells, the expression of microsomal CYP2R1 was considerably stronger.Moreover, the 25-hydroxylation activity of CYP2R1 is 26-fold higher than that of CYP27A1. 41This means ocular cells may be able to synthesize vitamin D 3 directly on exposure to sunlight.However, it should be noted that the American Academy of Ophthalmology recommends protection of the eye from long hours of direct sunlight, which may contribute to cataract and cancer formation.
The expression of megalin and cubilin in ocular barrier cells indicated the importance of these receptors in vitamin D 3 metabolism.Although megalin is expressed more widely in different tissues, the expression of cubilin is more pronounced in ocular barrier cells. 10,42n immune privileged organ, such as the eye, requires efficient strategies that can protect it from sight-threatening infections and damaging inflammatory responses.A recent study has shown that mice injected with vitamin D 3 had reduced retinal inflammation and this was associated with a reduction of retinal macrophage activation.More importantly, these mice showed decreased levels of amyloid-beta (Ab), a risk factor for age-related macular degeneration (AMD). 43Decreased serum levels in humans of 25(OH)D 3 have been correlated with increased early AMD and low visual acuity in the elderly. 44This was investigated further by calculating UVB exposure index, which correlated with reduced neovascular AMD.Moreover, single nucleotide polymorphisms of human CYP24A1 have been shown to be linked to neovascular AMD. 31 The ability of ocular barrier cells to locally produce large amounts of 1,25(OH) 2 D 3 may be important for their barrier function.For example, corneal endothelial cells are highly metabolic and represent an important barrier at the anterior segment of the eye, and producing large amounts of 1,25(OH) 2 D 3 may help maintain this function.More than 200 genes were significantly upregulated or downregulated in response to stimulation with 1,25(OH) 2 D 3 , including several involved in the immune response. 45he main caveat in this work is the use of cell lines rather than primary cells.However the lines used are well-established and regarded as good correlates for freshly-isolated ocular cells in multiple experiments.
In conclusion, this study has demonstrated for the first time to our knowledge the expression of vitamin D 3 synthesizing components in ocular barrier cells, and that these cells can convert inactive 25(OH)D 3 into active 1,25(OH) 2 D 3 .Further work will show if these cells can create an environment rich in 1,25(OH) 2 D 3 and if this has an effect on ocular immune protection and immune privilege.

FIGURE 1 .
FIGURE 1. Ocular barrier cells express mRNA for vitamin D 3 elements.Conventional RT-PCR was carried out using total RNA extracted from ocular barrier epithelial cell lines and two primary HSF samples (1 and 2), and primers for (A) VDR, CYP27B1, CYP24A1, CYP27A1, CYP2R1, and for (B) cubilin, and megalin.Ribosomal 18S was used as an internal PCR control and water was used a negative PCR control.The HKC-8 cells were used as a positive control for vitamin D machinery, and primary thyroid cells were used as a positive control for cubilin and megalin.Results are representative of at least three independent experiments.Molecular sizes (base pairs [bp]): 18S (400), VDR (421), CYP27B1 (302), CYP24A1 (485), CYP27A1 (292), CYP2R1 (259), cubilin (518), and megalin (290), and these show the constitutive expression of vitamin D 3 synthesizing components in ocular barrier cells.

FIGURE 2 .
FIGURE 2. Ocular barrier cells express proteins for vitamin D 3 elements.Ocular epithelial cell lines and primary HSF cells were grown to 40% to 50% confluency in 8-well chamber slides, then fixed and processed for immunofluorescence.Cells were stained with primary mouse monoclonal anti-human VDR antibody, mouse anti-human CYP24A1, and sheep polyclonal anti-human CYP27B1 antibodies, respectively.Staining was visualized with secondary FITC (green) conjugated anti-mouse and anti-sheep antibodies, respectively, using fluorescence microscopy.Nuclei were stained with DAPI (blue).This figure shows merged images with magnification: 320 for HSF and 340 for the others, and these show the expression patterns of vitamin D 3 synthesizing proteins in ocular barrier cells.Scale bars: 20 lm.

FIGURE 3 .
FIGURE 3. CYP27B1 co-localizes with M6P receptor and TGN38 in HCEC-12 .(A) Fluorescence microscopy images showing HCEC-12 cells grown to 40% to 50% confluency in 8-well chamber slides, then fixed and processed for immunofluorescence.Cells were stained with sheep polyclonal anti-human CYP27B1 and mouse monoclonal anti-human M6P and TGN38, respectively.Staining was visualized with FITC (green) and TR (red) conjugated anti-sheep and anti-mouse antibodies, respectively.Nuclei were stained with DAPI (blue).Results show colocalization of CYP27B1 with M6P (top) and TGN38 (middle).Negative control (bottom): cells with primary antibody omitted (bottom).Magnification: 3100 for M6P and TGN38 and 340 for negative control.Scale bars: 5 lm for M6P and TGN38, and 20 lm for negative control.(B) Confocal microscopy images showing HCEC-12 cells prepared as above and were stained with primary sheep polyclonal anti-human CYP27B1 antibodies.Staining was visualized with secondary FITC (green) conjugated anti-sheep antibodies.Nuclei were stained with DAPI (gray).Magnification: 340.Results show the perinuclear localization of CYP27B1 in HCEC-12 cells.

FIGURE 4 .
FIGURE 4. Ocular barrier cells can convert 25(OH)D 3 into active 1,25(OH) 2 D 3 .Ocular epithelial cells lines grown to 80% confluency in 12-well plates were either left untreated or treated with 25(OH)D 3 (10 À7 M) for 24 hours with or without pretreatment with CYP450 inhibitor (10 À5 M) ketoconazole (KC) for 2 hours.Cell culture supernatants were collected and 1,25(OH) 2 D 3 was determined by enzyme immunoassay.Results were obtained from a standard curve as pmol/L, then corrected per mg of protein.The HKC-8 cells were used as a positive conversion control.Results are representative for at least three independent experiments.Graph shows mean 6 SD 1,25(OH) 2 D 3 per mg protein for each treatment.Bottom right graph shows conversion rates in all ocular barrier epithelial cells compared to HKC-8.Kruskal-Wallis test with Dunn's posttest were performed (*P < 0.05, **P < 0.01; ns, not significant).Results show that all ocular barrier cells except HSF convert inactive to active 1,25(OH) 2 D 3 .

FIGURE 5 .
FIGURE 5.The rate of 25(OH)D 3 conversion by ocular barrier cells according to their location from the outside to the inside.A schematic diagram showing 1,25(OH) 2 D 3 concentration in the different ocular barrier cells from the exterior to the interior following their anatomic location.While no conversion was seen in HSF, 25(OH)D 3 conversion is high in the corneal epithelium (PHCEC), reaches the maximal levels in the corneal endothelium (HCEC-12), decreases in the ciliary body (ODM-2), and further in the retina (ARPE-19).

TABLE 1 .
Polymerase Chain Reaction Primer Sequence and Cycle Conditions 1,25(OH) 2 D 3 Enzyme Immunoassay (EIA)Cells were grown to confluence in 12-well culture plates.Cells were placed in serum-free medium and were either left untreated or treated with 25(OH)D 3 (10 À7 M) with or without pretreatment with ketoconazole (10 À5 M), an inhibitor of CYP27B1 (both Sigma-Aldrich) for 2 hours.After 24 hours, cell culture supernatants were collected for the measurement of 1,25(OH) 2 D 3 , and cells were processed for protein extraction and quantification.The 1,25(OH) 2 D 3 concentration was measured by EIA (Immunodiagnostics Systems Limited, Tyne & Wear, UK) according to the manufacturer's instructions.conjugated anti-mouse or FITC conjugated anti-sheep secondary antibody (The Binding Site Ltd.), or Texas Red (TR) conjugated anti-mouse antibody (Abcam) diluted 1:250 in blocking solution for 1 hour at room temperature.Cells were counterstained briefly with DNA-specific stain DAPI (Gibco, Invitrogen) and mounted (Vectashield; Vector Laboratories, Peterborough, UK).Staining was detected using a fluorescent microscope (AxioPlan2 Imaging; Zeiss, Cambridge, UK).

TABLE 2 .
A Summary of Gene and Protein Expression for Vitamin D 3 Elements by Ocular Barrier Cells as Well as the Rate of 25D 3 Conversion