Forkhead Box Transcription Factors of the FOXA Class Are Required for Basal Transcription of Angiotensin-Converting Enzyme 2

Figures

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  Figure 1.. The proximal ACE2 promoter is active in 832/13 cells and pancreatic islets. (a) Luciferase reporters containing human ACE2 promoter segments were transfected into 832/13 cells. A putative ERE (put ERE) is indicated. Transfected cells were treated with 0 or 100 nM estradiol (E2) for 44 hours. (b) Luciferase reporters containing human ACE2 promoter segments were transfected into 832/13 cells together with 0.5 μg EGFP-ERα and 0.5 μg EGFP-ERβ expression plasmid per well. A put ERE is indicated. Transfected cells were treated with 0 or 100 nM estradiol (E2) for 44 hours. ***P < 0.001 vs 0 nM E2. (c) 832/13 cells were transfected with EGFP-ERα and 0.5 μg EGFP-ERβ expression plasmids and subsequently treated with 0 or 100 nM E2 for 48 hours in four experiments. The content of ACE2 mRNA relative to β-actin mRNA was determined. (d) An EMSA was done with the probes for the put EREs from the mouse and human ACE2 promoters as well as the ERE from the vitellogenin A2 gene. Nuclear extracts were from 832/13 cells that were untransfected or transfected with either EGFP-ERα or EGFP-ERβ expression plasmids and grown in the presence of 100 nM E2. (e) In mouse pancreatic islets, the concentration…

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  Figure 2.. Identification of cis-regulatory elements controlling ACE2 expression. (a) The conserved regions of the human proximal ACE2 promoter region and bases that were mutated in luciferase reporters by in vitro mutagenesis are indicated. Pink shading indicates bases conserved among seven placental mammalian species (human, mouse, rabbit, dog, panda, horse, and cow), and blue shading indicates the introduced mutations of 14 regions (R1 to R14). (b) 832/13 cells were transfected with luciferase reporters containing wild-type or mutated sequences of the human ACE2 proximal promoter region. Luciferase activities were determined 44 hours after transfection. R3, R4, R6, R8, and R12 denote the promoter regions, where mutations significantly affected luciferase activities. ***P < 0.001 vs the unmutated, wild-type −454/−1 promoter sequence. (c) An EMSA was conducted with nuclear extracts from 832/13 cells and with DNA probes that were wild-type and mutated sequences of five regions with putative cis-regulatory elements.

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  Figure 3.. Forkhead box transcription factors bind to the ACE2 proximal promoter region. (a) An EMSA was conducted with the R4 DNA probe. Nuclear extracts were from untransfected 832/13 cells or 832/13 cells transfected with COUP-TFII or PPARγ expression plasmids. Antibodies against COUP-TFII and PPARγ were included in the binding reactions as indicated. (b) The R6 region has similarity to FOXO1 and FOXA1 motifs as indicated by the BKL TRANSFAC program, whereas the mutation destroys the similarity. (c) An EMSA was done with the R6 probe and nuclear extracts from 832/13 cells that were untransfected or transfected with a FOXO1 expression plasmid (left panel). The right panel shows the effect on the band pattern when an antibody against FOXO1 is included. (d) An EMSA was done with the R6 probe and nuclear extracts from 832/13 cells that were untransfected or transfected with FOXA1, FOXA2, or FOXA3 expression plasmids (left panel). The right panel shows the effects on the band pattern when antibodies against the FOXA transcription factors are included in the binding reactions. (e) An EMSA was conducted with a nuclear extract from 832/13 cells. The probes were the human and mouse R6 regions as…

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  Figure 4.. FOXA transcription factors stimulate ACE2 expression. (a) 832/13 cells were transfected with luciferase reporters containing the wild-type human −454/−1 ACE2 promoter sequence or the promoter sequence in which the FOXA motif was mutated. Cells were cotransfected with expression plasmids for FOXA1, FOXA2, FOXA3, or a control plasmid. Luciferase activities were determined 44 hours after transfection and corrected for the background activities of empty pGL3-Basic. *P < 0.05, **P < 0.01 vs control. (b) 832/13 cells were transfected with expression plasmids for forkhead box transcription factors or a control plasmid. RNA was isolated 22 hours after transfection. The content of ACE2 mRNA relative to β-actin mRNA was determined. ***P < 0.001 vs control. (c) 832/13 cells were transfected with luciferase reporters containing the wild-type human −454/−1 ACE2 promoter sequence or the promoter sequence in which the FOXA motif was mutated. Transfected cells were treated with 0.5 mM AICAR or dimethyl sulfoxide (DMSO) vehicle as control for 44 hours. Luciferase activities were corrected for the background activities of empty pGL3-Basic. *P < 0.05, ***P < 0.001 vs control. (d) 832/13 cells were…

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  Figure 5.. FOXA2 is the major protein in mouse islets binding the FOXA motif in the proximal ACE2 promoter. (a) An EMSA with the human R6 probe was conducted using nuclear extracts from mouse pancreatic islets and 832/13 cells. (b) An EMSA with the mouse R6 probe was conducted using cytoplasmic and nuclear extracts from mouse pancreatic islets. Antibodies against FOXA transcription factors were included in binding reactions as indicated. (c) The concentrations of FOXA2 mRNA and ACE2 mRNA relative to 18S ribosomal RNA were determined in freshly isolated pancreatic islets from six male and five female mice. We depict the ACE2 values against the FOXA2 values for each mouse. The solid and stippled lines are the regression lines for females and males, respectively. The concentrations of FOXA2 and ACE2 showed significant (P < 0.01) correlation for both males and females as indicated by the correlation coefficients R. (d) The content of ACE2 mRNA relative to FOXA2 mRNA was calculated. *P < 0.05 vs males.