Foxo Regulates Expression Of Transporter Protein ABCA6

Objectives:ATP binding cassette (ABC) transporters is a transmembrane protein.It can mediate transmembrane transport of lipid and other molecules via ATP. Human ABC transporter protein includes A-G subfamilies. ABCA6is one of ABCA subfamily members, but its transcriptional regulation and physiological function remain unknown so far. In a microarray analysis of expression profile in human endothelial cell line transfected with FoxO plasmids, we found that overexpression of the transcription factor FoxO results in up-regulation of ABCA6. In the current study, we investigated the mechanism of FoxO regulating ABCA6expression and explored the function of ABCA6preliminarily.Methods:Cells were transfected with FoxO expression plasmids or FoxO siRNA respectively, ABCA6mRNA was then quantified using real-time PCR. The human ABCA6reporter plasmid was generated by subcloning the promoter about800bp upstream of the start of cDNA. Cells were co-transfected with reporter plasmid and either a control or a FoxO-expressing plasmid and luciferase activity was measured; To identify the binding sites of FoxO in ABCA6promoter, a series of deletion or mutation constructs were generated, and reporter assay was performed. EMSA and CHIP were further employed to confirm the binding of FoxO to the ABCA6promoter both in vitro and in vivo. Moreover, ABCA6levels were examined by real-time PCR and western blots during glucose deprivation. To further study the fuction of ABCA6expression, we constructed ABCA6expression plasmid and builded cells of stable transfection ABCA6. Through the fluorescence immunohistochemistry and confocal microscopy, we observated that ABCA6locates in intracellular and detected lipid metabolism related gene Insigl mRNA using real-time PCR.Results:Transfection with constitutively active FoxO1TM or FoxO3aTM, led to significantly increase of ABCA6mRNA in human endothelial cells; Depletion of either FoxO1or FoxO3a was associated with reduction of ABCA6mRNA. To the ABCA6promoter analysis by rVista, we find that the two binding sites of FoxO may be identified. We observed dramatic increase of ABCA6promoter activity with FoxO1, FoxO3, FoxO4co-expression. Deletion or mutation of any binding site significantly suppressed the activition ABCA6promoter to FoxO. EMSA results exhibited a nuclear extract prepared from cells transfected with FoxO overexpression plasmids had obviously bingding with the ABCA6promoter. CHIP further demonstrate that the DNA fragment could be amplified by PCR in the presence of antibody. We also find intracellular FoxO is significantly decreased during glucose deprivation, and ABCA6mRNA is also significantly reduced. In addition, ABCA6locating Golgi apparatus may involve in the cholesterol transport and metabolism.Conclusion:We here demonstrated that expression of ABCA6was FoxO-dependent in human endothelial cells. The FoxO can activate significantly ABCA6promoter. FoxO directly regulates ABCA6transcription by combinating two sites of ABCA6promoter. During glucose deprivation, intracellular FoxO is decreased and ABCA6mRNA is also significantly reduced. ABCA6is localized primarily in Golgi apparatus, and may participate in intracellular cholesterol transport and metabolism.