1?,25-Dihydroxyvitamin D3 Up-regulates Interleukin-34 Expression In SH-SY5Y Neural Cells

Background & Objective: Vitamin D supplements are regarded as a novel approach to reduce the pathologic symptoms of Alzheimer's disease.But its mechanism is not completely known.The active form of vitamin D,calcitriol or 1?,25-Dihydroxyvitamin D3(1?,25(OH)2D3),mediates a variety of biological actions by binding to the cytoplasmic vitamin D receptor(VDR).IL-34 provides strong neuroprotective and survival signals in brain injury and neurodegeneration.Amyloid-? peptide(A?)is a key molecule in the pathogenesis of Alzheimer's disease(AD).IL-34 promotes microglial proliferation and clearance of soluble oligomeric amyloid-?(o A?)in AD.IL-34 increases the expression of insulin-degrading enzyme,aiding the clearance of o A?,and induces the antioxidant enzyme heme oxygenase-1 and TGF-? in microglia to reduce oxidative stress.It was reported that 1?,25(OH)2D3 up-regulated mouse IL-34 m RNA levels in osteoblastic cells.The aim of this study is to investigate whether human IL-34 is up-regulated by 1?,25(OH)2D3 in neural cells.Methods:Endogenous expression of IL-34 in a variety of cell lines(Hep G2,SH-SY5 Y,A172,GES-1,SGC-7901,HGC-27)were detected using Western blot.SH-SY5 Y cells were treated with increasing concentrations of 1?,25(OH)2D3 for 72 h;SH-SY5 Y cells were incubated with 100 n M 1?,25(OH)2D3 for the different time intervals,then IL-34 expression was analyzed by Western blot.For identification of IL-34 core promoter,four DNA fragments containing the upstream regions of the IL-34 gene were amplified and cloned into p GL3-Basic luciferase reporter plasmids.SH-SY5 Y and GES-1 cells were transfected with the IL-34 promoter constructs respectively,IL-34 core promoter region was analyzed by luciferase assay.In order to elucidate how 1?,25(OH)2D3 induced IL-34 expression in SH-SY5 Y cells,we transfected p GL3-713,p GL3-475 and p GL3-326 into SH-SY5 Y cells,cells were subsequently treated with 1?,25(OH)2D3.SH-SY5 Y cells were co-transfected with p GL3-326 and VDR si RNA or control si RNA respectively and cells were harvested for Western blot and Luciferase assay after further stimulation.IL-34 upstream DNA sequence is analyzed by PATCH on the web site of www.gene-regulation.com.Three possible Vitamin D receptor binding sites are predicted.The p GL3-326 mutants with no VDR binding sites were constructed and transfected into SH-SY5 Y cells respectively for luciferase assay.Results:1.IL-34 was detectable in a variety of cell lines.2.IL-34 expression was up-regulated by 1?,25(OH)2D3 in a dose-and time-dependent manner in SH-SY5 Y cells.3.The core promoter of IL-34 gene was located within the region-274/-3 relative to the transcription start site.4.1?,25(OH)2D3 induced IL-34 expression through VDR in SH-SY5 Y cells.5.A VDR binding site(CGCCCT)was required for 1?,25(OH)2D3-induced IL-34 expression in SH-SY5 Y cells.Conclusion:1?,25(OH)2D3,the active form of Vitamin D,induced IL-34 expression in SH-SY5 Y neural cells.