Fig. 1.

Up-regulation of BACE1 gene transcription by hypoxia. (A) Hypoxia increases human BACE1 promoter activity. BACE1 promoter constructs pB1P-H and pB1P-I were transfected into SH-SY5Y cells. Plasmid pGL3-Basic (vector) and pEpoE-Luc were used as negative and positive controls, respectively. Cells were exposed to 2% O₂ (hypoxia) or 21% O₂ (control) after transfection. Luciferase assay was performed 48 h after transfection to reflect promoter activity. ∗, P < 0.001 by ANOVA and Student's t test. (B) HIF-1α expression was rapidly induced by hypoxia in SH-SY5Y cells. Cells were treated with 2% O₂ for 0, 1, and 2 h and then lysed in RIPA-Doc buffer (0.15 mM NaCl/0.05 mM Tris · HCl, pH 7.2/1% Triton X-100/1% sodium deoxycholate/0.1% SDS). HIF-1α was detected by rabbit polyclonal anti-HIF-1α antibody (H206). (C) Diagram shows base pairs −980 to −900 of the human BACE1 promoter sequence (relative to the transcription start site). A HRE consensus site 5′-RCGTG was located at base pairs −915 to −911 (capitalized and underlined). (D) The human BACE1 promoter contains a HRE site. Gel shift assay was performed as described in Methods. A ³²P-labeled double-stranded oligonucleotide probe, [³²P]BACE1-HRE, corresponding to BACE1 promoter base pairs −924 to −907 was used as a probe. (E) Robust HIF-1α expression in HEK293 cells after pHIF-1α expression plasmid transfection. (F) BACE1 promoter activity was increased by HIF-1α overexpression. BACE1 promoter constructs pB1P-H, pB1P-I, and positive control pEpoE-Luc were cotransfected with HIF-1α expression plasmid into HEK293 cells. Luciferase assay was performed 48 h after transfection. HIF-1α overexpression can significantly increase promoter activity in cells transfected with pB1P-H but not pB1P-I. HIF-1α overexpression also significantly increased pEpoE-Luc promoter activity. ∗, P < 0.001 by ANOVA. (G) HIF-1α siRNA transfection reduced HIF-1α expression in HEK293 cells. (H) HIF-1α siRNA inhibited hypoxia's up-regulatory effect on the human BACE1 promoter activity. BACE1 promoter construct pB1P-H or positive control pEpoE-Luc plasmid were cotransfected with control siRNA or HIF-1α siRNAs. The transfected cells were then exposed to 2% O₂ (hypoxia) or 21% O₂ (control) 12 h after transfection. Luciferase assay was performed 24 h after hypoxia treatment to reflect promoter activity. pCMV-Rluc was cotransfected to normalize transfection efficiency. The numbers represent mean ± SEM; n = 4; ∗, P < 0.0001 by Student's t test. (I) Mutation in the HRE site abolishes the effect of hypoxia on BACE1 promoter activity. The HRE site in the BACE1 promoter of pB1P-H was mutated by site-directed mutagenesis. The mutant construct was transfected into SH-SY5Y cells and exposed to 2% O₂ (hypoxia) or 21% O₂ (normoxia) for 48 h. (J) Swedish APP stable SH-SY5Y cells were exposed to 2% or 21% O₂ for 24 h before RNA extraction. (K) Hypoxia increased BACE1 mRNA by ≈1.5 times. ∗, P < 0.05 by Student's t test.