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Xiaowen Lu, Mitchell A Watsky; Effects of 1,25-Vitamin D3 and24,25-Vitamin D3 on Corneal Epithelial Gap Junctions. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5624.
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To study the effects of 1,25-VitD3 and 24,25-VitD3 on corneal epithelial cell gap junctions.
Real-time PCR was used to determine the mRNA expression of connexins 26, 30, and 43 in a human corneal epithelial cell line (HCEC). HCEC were treated with 10 nM 1,25-VitD3 or 50 nM 24,25-VitD3 and mRNA was obtained at 24 hours. Western blotting was used to detect gap junction proteins. Real-time PCR and western-blot were also used to detect mRNA and protein expression of gap junctional proteins in HCEC following siRNA-mediated VDR silencing. In vitro gap juntion function was assessed in HCEC using the scrape loading/dye transfer assay and in situ gap junction diffusion coefficients of corneal epithelial and limbal cells were measured using fluorescence recovery after photobleaching (FRAP). FRAP studies were performed on 5(6)-carboxyfluorescein diacetate-stained mouse corneas and corneal limbus.
Connexin 26, 30, and 43 mRNA levels were significantly increased in HCEC treated with both 1,25-VitD3 and 24,25-VitD3. The mRNA level of the gap junction proteins were decreased in HCEC when VDR was silenced with siRNA. The connexin 43 protein level was significantly increased in HCEC cultured with 1,25-VitD3 and 24,25-VitD3. Gap junction connectivity was significantly increased in HCEC cultured with 50 nM 24,25-VitD3 (P<0.01, n=5) as determined using the scrape loading/dye transfer assay while 10 nM 1,25-VitD3 had no significant effect on gap junction connectivity. The FRAP assay determined that gap junction diffusion coefficients in corneal limbal cells (23.76±1.63) is significantly higher than in corneal epithelial cells (18.71± 1.98) (P<0.01).
Both 1,25-VitD3 and 24,25-VitD3 increase mRNA levels of connexins 26, 30 and 43 and the connexin 43 protein level in HCEC. 24,25-VitD3 also increases HCEC intracellular connectivity. We also found that mouse corneal limbal cells have higher gap junction diffusion coefficients than central epithelial cells.
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