Transcriptional regulation of the human CYP46A1 brain‐specific expression by Sp transcription factors

Abstract

Brain defective cholesterol homeostasis has been associated with neurologic diseases, such as Alzheimer's and Huntington's disease. The elimination of cholesterol from the brain involves its conversion into 24(S)-hydroxycholesterol by CYP46A1, and the efflux of this oxysterol across the bloodbrain barrier. Herein, we identified the regulatory elements and factors involved the human CYP46A1 expression. Functional 5¢deletion analysis mapped a region spanning from nucleotides -236/-64 that is indispensable for basal expression of this TATA-less gene. Treatment of SH-SY5Y cells with mithramycin A resulted in a significant reduction of promoter activity, suggesting a role of Sp family of transcription factors in CYP46A1 regulation. Combination of Sp1, Sp3, and Sp4 over-expression studies in Drosophila SL-2 cells, and systematic promoter mutagenesis identified Sp3 and Sp4 binding to four GC-boxes as required and sufficient for high levels of promoter activity. Moreover, Sp3 and Sp4 were demonstrated to be the major components of the protein-DNA complexes observed in primary rat cortical extracts. Our results suggest that the cell-type specific expression of Sp transcription factors -substitution of Sp1 by Sp4 in neurons -is responsible for the basal expression of the CYP46A1 gene. This study delineates for the first time the mechanisms underlying the human CYP46A1 transcription and thereby elucidates potential pathways underlying cholesterol homeostasis in the brain.

Transcriptional regulation of cholesterol 24-hydroxylase by histone deacetylase inhibitors

“…However, there are several examples in the literature indicating that CYP46A1 can be induced [10][11][12]. A recent study by Milagre et al indicated that members of the SP1 family of transcription factors might regulate CYP46A1 [13].…”

Cholesterol 24S-Hydroxylase Overexpression Inhibits the Liver X Receptor (LXR) Pathway by Activating Small Guanosine Triphosphate-Binding Proteins (sGTPases) in Neuronal Cells

“…Therefore, we started to investigate whether an increase in CYP46A1 expression affects prenylation and activity of members of the Rho and Rab families of sGTPases in two different cellular models, with distinct endogenous CYP46A1 expression levels. We have used primary cultures of rat cortical neurons maintained for 26DIV which express high levels of CYP46A1, and SH-SY5Y human neuroblastoma cells, where CYP46A1 mRNA can barely be detected [43]. Firstly, to determine if the recombinant CYP46A1 protein retains its catalytic activity, we have transfected SH-SY5Y cells and 26DIV neurons with an expression vector coding for the human CYP46A1 (pFLAG-hCYP46A1) for 48 and 24 h, respectively.…”

“…The SH-SY5Y human neuroblastoma cell line was cultured as previously described [43]. Briefly, cells were maintained in low glucose DMEM (Sigma), at 37°C in humidified 5 % CO 2 .…”

Okadaic acid inhibits the trichostatin A-mediated increase of human CYP46A1 neuronal expression in a ERK1/2-Sp3-dependent pathway

“…The different recombinant wild-type and mutated plasmids derived from the 5 ′ fl anking region of the human CYP46A1 gene and used in this work have also been described previously ( 12 ). NTERA-2cl.…”

“…The SH-SY5Y human neuroblastoma cell line was maintained and transiently transfected as previously described ( 12 ). The different recombinant wild-type and mutated plasmids derived from the 5 ′ fl anking region of the human CYP46A1 gene and used in this work have also been described previously ( 12 ).…”