Vitamin D Regulation of CD14 in Mammary Epithelial Cells

描述（由申请人提供）：目前的项目将研究维生素D在乳腺上皮细胞中对先天免疫蛋白CD14的调节。虽然维生素D在免疫细胞中的作用已经得到了很好的认识，但维生素D对上皮细胞免疫反应的影响尚未得到很好的研究。人乳腺上皮细胞（hTERT-HME）的初步数据表明，维生素D诱导CD14的表达和分泌，CD14是一种结合细菌脂多糖（LPS）的蛋白质。本研究将阐明维生素D对乳腺上皮细胞CD14和先天免疫反应的影响。核心假设是，维生素D通过VDR促进抗炎环境，刺激可溶性CD14从乳腺上皮细胞释放。在目的1中，我们将确定是否需要VDR来诱导CD14合成以响应生理剂量的维生素D，如果是，VDR信号在细胞核和质膜中的相对贡献。为此，我们将在具有shRNA结构的hTERT- HME细胞中敲除VDR，并利用维生素D类似物选择性地激活核或膜VDR活性。结果将包括qPCR检测CD14基因表达和ELISA检测sCD14分泌。在Aim 2中，我们将评估维生素D是否会改变hTERT-HME细胞中膜CD14 （mCD14）的表达，并表征响应维生素D分泌的可溶性CD14 （sCD14）的亚型。这些细胞将用浓度为1,25D （VDR配体）或25D（模拟人体生理循环浓度的循环维生素D代谢物）处理。结果将包括显微镜检查mCD14表达和western blotting鉴定特定的sCD14亚型。由于mCD14通过糖基磷脂酰肌醇（GPI）锚定锚定在膜上，我们将研究基因组抑制GPI合成是否会阻断维生素d的作用。我们还将使用化学抑制剂阻断分泌途径，以确定其对维生素d诱导的sCD14的影响。在Aim 3中，我们将研究乳腺细胞释放的sCD14对维生素d的反应功能。我们将测试由维生素d处理的hTERT-HME细胞衍生的条件培养基对THP-1巨噬细胞免疫反应的影响。THP-1细胞将在条件培养基中孵育，并用LPS攻毒。采用qPCR和ELISA检测THP-1细胞促炎和抗炎细胞因子的表达和分泌情况。我们还将通过流式细胞术确定维生素d诱导的sCD14是否会改变巨噬细胞对LPS的结合或释放。我们预计，拟议的研究将证明维生素D触发乳腺上皮细胞分泌sCD14并促进抗炎环境。我们将定义25D在抗炎反应方面的剂量反应，这将有助于了解维生素D缺乏或维生素D过量是否会改变体内的这些反应。总的来说，这项工作将增强我们对维生素D在上皮组织中的免疫功能的理解。

DESCRIPTION (provided by applicant): The current project will investigate the regulation of an innate immunological protein, CD14, by vitamin D in mammary epithelial cells. Although actions of vitamin D in immune cells are well recognized, effects of vitamin D on the immune responses of epithelial cells have not been well studied. Preliminary data with human mammary epithelial (hTERT-HME) cells demonstrated that vitamin D induces expression and secretion of CD14, a protein that bind bacterial lipopolysaccharide (LPS). The proposed study will clarify the effect of vitamin D on CD14 and the innate immune response in mammary epithelial cells. The central hypothesis is that vitamin D promotes an anti-inflammatory environment through VDR to stimulate of soluble CD14 release from mammary epithelial cells. In Aim 1 we will determine if VDR is required for the induction of CD14 synthesis in response to physiological doses of vitamin D and, if so, the relative contributions of VDR signaling in the nucleus and at the plasma membrane. For this aim we will knock down VDR in the hTERT- HME cells with shRNA constructs and utilize vitamin D analogs that selectively activate nuclear or membrane VDRs activity. Outcomes will include CD14 gene expression by qPCR and sCD14 secretion by ELISA. In Aim 2 we will assess whether vitamin D alters the expression of membrane CD14 (mCD14) and characterize the isoforms of soluble CD14 (sCD14) secreted in response to vitamin D in hTERT-HME cells. These cells will be treated with concentrations of 1,25D (the VDR ligand) or 25D (the circulating vitamin D metabolite that will mimic physiological circulating concentrations in humans). Outcomes will include microscopy for mCD14 expression and western blotting to identify specific sCD14 isoforms. Because mCD14 is tethered to the membrane by a glycosylphosphatidylinositol (GPI) anchor, we will examine whether genomically inhibiting GPI synthesis blocks the effects of vitamin D. We will also use a chemical inhibitor to block the secretory pathway to determine its impact on vitamin D-induced sCD14. In Aim 3 we will investigate the functionality of sCD14 released from mammary cells in response to vitamin D. We will test the effects of conditioned media derived from vitamin D-treated hTERT-HME cells on immune responses of THP-1 macrophages. The THP-1 cells will be incubated in the condition media and challenged with LPS. The expression and secretion of pro- and anti- inflammatory cytokines by THP-1 cells will be assessed by qPCR and ELISA. We will also determine if the vitamin D-induced sCD14 alters binding or release of LPS from the macrophage by flow cytometry. We anticipate that the proposed studies will demonstrate that vitamin D triggers mammary epithelial cells to secrete sCD14 and promotes an anti-inflammatory environment. We will define the dose response of 25D with respect to anti-inflammatory responses, which will provide insight into whether vitamin D deficiency or vitamin D excess might alter these responses in vivo. Overall this work will enhance our understanding of the immunological functions of vitamin D in epithelial tissues.