Effect of Vitamin D Receptor Knockout on Cornea Epithelium Wound Healing and Tight Junctions

PURPOSE. Our laboratory previously determined that vitamin D3, the vitamin D receptor (VDR), and 1a hydroxylase are present and active in the eye. In this study, we examined the effects of VDR knockout on wound healing, the tight junction-associated proteins occludin and ZO-1, and tight junction numbers in mouse corneas. METHODS. Epithelial wounds (2-mm) were made with an agar brush on 4-week-old and 10-week-old wild-type, heterozygous, and VDR knockout mouse corneas. Mice were on a normal or high lactose, Ca 2þ , and PO 4 À diet. Wound-healing area was measured over time. Real-time PCR was used to quantify occludin and ZO-1 message expression. Western blot was used for protein expression. Transmission electron microscopy was used to examine corneal epithelium and endothelium tight junctions. Immunofluorescence was used to examine epithelial ZO-1 distribution. RESULTS. Results showed a decreased healing rate in 10-week-old VDR knockout mice compared with wild-types. Vitamin D receptor knockout mice on the special diet had no difference in healing rate compared with wild-types. Real-time PCR showed decreased expression of occludin and ZO-1 in 10-week-old VDR knockout mice compared with wild-types. Western blot of 10-week-old knockout mouse corneas showed decreased occludin expression compared with wild-types. Transmission electron microscopy showed a significant difference in tight junction numbers in VDR knockouts versus wild-types. Immunofluorescence showed a change in ZO-1 distribution among genotypes. CONCLUSIONS. Vitamin D receptor knockout affects mouse corneal epithelium wound healing and tight junction integrity

PPIP5K2 and PCSK1 are Candidate Genetic Contributors to Familial Keratoconus

Keratoconus (KC) is the most common corneal ectatic disorder affecting >300,000 people in the US. KC normally has its onset in adolescence, progressively worsening through the third to fourth decades of life. KC patients report significant impaired vision-related quality of life. Genetic factors play an important role in KC pathogenesis. To identify novel genes in familial KC patients, we performed whole exome and genome sequencing in a four-generation family. We identified potential variants in the PPIP5K2 and PCSK1 genes. Using in vitro cellular model and in vivo gene-trap mouse model, we found critical evidence to support the role of PPIP5K2 in normal corneal function and KC pathogenesis. The gene-trap mouse showed irregular corneal surfaces and pathological corneal thinning resembling KC. For the first time, we have integrated corneal tomography and pachymetry mapping into characterization of mouse corneal phenotypes which could be widely implemented in basic and translational research for KC diagnosis and therapy in the future

Vitamin D in Tear Fluid

PURPOSE. To determine the source(s) of vitamin D in tear fluid and examine the expression of the endocytic proteins and putative vitamin D transporters megalin and cubilin in lacrimal and Harderian glands. METHODS. Wild-type, heterozygous, and vitamin D receptor (VDR) knockout C57BL/6 mice were used, with a subset of knockout mice fed a replenishment diet for some studies. Mouse lacrimal and Harderian glands from each group were used to measure megalin and cubilin by RT-PCR, Western blot, and immunohistochemistry. New Zealand white rabbits were used to collect lacrimal and accessory gland fluid for vitamin D mass spectroscopy measurements. RESULTS. Ten-week-old knockout mice were significantly (P < 0.05) smaller than wild-type mice. Real-time PCR and Western blot showed decreased expression of megalin and cubilin in select VDR knockout mouse groups. Immunohistochemistry showed apical duct cell megalin staining and weaker megalin staining in VDR knockout mice compared with controls. Vitamin D2 was more prevalent in rabbit lacrimal and accessory gland fluid than vitamin D3, and greater amounts of Vitamin D2 were found in in tear fluid obtained directly from lacrimal and accessory glands as compared with plasma concentrations. CONCLUSIONS. This is the first study to demonstrate the presence of megalin and cubilin in lacrimal and accessory glands responsible for producing tear fluid. The results strengthen the hypothesis that megalin and cubilin are likely involved in the secretory pathway of vitamin D into tear fluid by the duct cells. Keywords: tears, vitamin D, megalin, cubilin, lacrimal gland N umerous studies have been published describing the nonclassical actions of vitamin D. Our lab discovered that the cornea is capable of activating and metabolizing vitamin D 1 This ability of the cornea to metabolize vitamin D was recently confirmed. 14 Cubilin is also an endocytic receptor that has been shown to be coexpressed with megalin in several absorptive epithelia and also has the potential for binding DBP. 3 The purpose of this study was to determine the source(s) of vitamin D in tear fluid. This is particularly relevant given a recent study demonstrating that high serum vitamin D levels have a small but favorable effect on dry eye syndrome. METHODS Animals Wild-type (þ/þ), heterozygous (þ/À), and VDR knockout (À/À) C57BL/6 mice were obtained and bred from the Jackson Labs

<i>Ex vivo</i> human corneal epithelial cell calcium wave video, DMEM, 20X objective.

Still photos in Fig 1J–1L captured from this video. Circle highlights the TPMD target on the source cell. (ZIP)</p

Primary mouse corneal epithelial cell calcium wave following thapsigargin exposure video, 20X objective.

Still photos in Fig 6E and 6F captured from this video. Circle highlights the TPMD target on the source cell. (ZIP)</p

Influence of thapsigargin and AMG9810 treatment on TPMD-Ca⁺⁺ Wvs compared to control DMEM in mouse and human CECs.

Representative images of primary mouse and human CECs are presented showing cells before and after laser induced TPMD initiation. White arrows show the TPMD source cell in each image. No other cells were wounded.</p

Primary human corneal epithelial cell calcium wave video, DMEM, 20X objective.

Still photos in Fig 1D–1F captured from this video. Circle highlights the TPMD target on the source cell. (ZIP)</p