Department of Cellular Biology and Anatomy, Medical College of Georgia, Augusta University, Augusta, GA, USA.
Department of Cellular Biology and Anatomy, Medical College of Georgia, Augusta University, Augusta, GA, USA; The Graduate School, Augusta University, Augusta, GA, USA.
Exp Eye Res. 2019 Oct;187:107776. doi: 10.1016/j.exer.2019.107776. Epub 2019 Aug 26.
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)D3 and 24R,25(OH)D3 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)D3 and 24R,25(OH)D3 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)D3 or 24R,25(OH)D3. Western blotting was used to detect gap junction proteins. Vitamin D3 effects on epithelial intracellular Ca (Ca) 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)D3 and 24R,25(OH)D3. 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)D3 and 1,25(OH)D3. Ca was not affected by 1,25(OH)D3 or 24R,25(OH)D3 in HCEC or MPCEC. We conclude that both 1,25(OH)D3 and 24R,25(OH)D3 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 Ca.
维生素 D 是一种脂溶性前激素，可在全身和个别组织中被激活。我们的实验室之前已经证明，角膜上皮可以激活维生素 D，维生素 D 代谢物 1,25(OH)D3 和 24R,25(OH)D3 可以影响角膜上皮细胞的迁移、增殖以及紧密和间隙连接功能。这些维生素 D 衍生的代谢物通过维生素 D 受体 (VDR) 传递信号。本研究的目的是专门确定 1,25(OH)D3 和 24R,25(OH)D3 对角膜上皮细胞间隙连接蛋白的影响。在人角膜上皮细胞系 (HCEC)、野生型和维生素 D 受体敲除 (VDR) 小鼠角膜以及培养的小鼠原代上皮细胞 (MPCEC) 中检测连接蛋白 (Cx) 26、30 和 43 的蛋白表达。通过划痕加载/染料转移测定评估体外间隙连接功能。用 1,25(OH)D3 或 24R,25(OH)D3 处理 HCEC 和 MPCEC。使用 Western blot 检测间隙连接蛋白。使用染料 Cal-520 测定上皮细胞内 Ca（Ca）对维生素 D3 的影响。用 1,25(OH)D3 和 24R,25(OH)D3 处理 HCEC 和 MPCEC 后，Cx26 和 Cx43 蛋白水平显著增加。VDR MPCEC 中 Cx30 和 Cx43 蛋白水平也显著增加。在培养有 24R,25(OH)D3 和 1,25(OH)D3 的 HCEC 和 MPCEC 中，体外间隙连接连接性显著增强。1,25(OH)D3 或 24R,25(OH)D3 对 HCEC 或 MPCEC 中的 Ca 没有影响。我们得出结论，1,25(OH)D3 和 24R,25(OH)D3 都是角膜上皮细胞连接蛋白和间隙连接通讯的正调节剂。这些维生素 D 代谢物似乎通过 VDR 依赖和非依赖途径传递信号。维生素 D 对角膜上皮细胞间隙连接的影响似乎不依赖于 Ca。
