Journal: Biochimica et biophysica acta

Article Title: The transcription factor HNF1α induces expression of angiotensin-converting enzyme 2 (ACE2) in pancreatic islets from evolutionarily conserved promoter motifs

doi: 10.1016/j.bbagrm.2013.09.007

Figure Lengend Snippet: HNF1α and HNF1β bind to three motifs in the proximal ACE2 promoter region. (A) EMSA with nuclear extracts from 832/13 cells transfected with the HNF1β expression plasmid or the control plasmid pRcNhe. The probes represent the 3 potential HNF1 binding sites in the proximal promoter region (1st, 2nd, and 3rd motif), the potential HNF1binding site in the distal promoter region (4th motif), and the HNF1 binding site from the rat β-fibrinogen promoter. Complex I is a complex generated for cells transfected with the HNF1β expression plasmid. An HNF1β antibody supershifts complex I (right panel). (B) EMSA with nuclear extracts from 832/13 cells transfected with the HNF1α expression plasmid or the control plasmid pCVM6-XL5. Complex II and II are complexes whose intensities increase for extracts from cells transfected with the HNF1α expression plasmid. The endogenous complexes II and III are supershifted with an HNF1 antibody (right panel). (C) EMSA with nuclear extracts from untransfected 832/13 cells. Complex IV is a fast-migrating complex binding to the 1st and 4th motif that can be supershifted with a GATA4 antibody.

Article Snippet: An expression plasmid for human HNF1α (cat. no. SC300093) and a corresponding empty control plasmid pCVM6-XL5 were purchased from OriGene Technologies, Inc., Rockville, MD.

Techniques: Transfection, Expressing, Plasmid Preparation, Binding Assay, Generated

Journal: Biochimica et biophysica acta

Article Title: The transcription factor HNF1α induces expression of angiotensin-converting enzyme 2 (ACE2) in pancreatic islets from evolutionarily conserved promoter motifs

doi: 10.1016/j.bbagrm.2013.09.007

Figure Lengend Snippet: HNF1α and HNF1β induce ACE2 expression in insulinoma cells. (A) Concentrations of ACE2 mRNA normalized to β-actin mRNA were determined for 832/13 cells and βTC3 cells transfected with the HNF1α expression plasmid, the HNF1β expression plasmid, or the corresponding control plasmids. Each panel summarizes results for four independent transfection experiments. (B) Western blotting was performed for 832/13 cells transfected with the HNF1α expression plasmid, the HNF1β expression plasmid, or the corresponding control plasmids. Each blot shows expression of ACE2 and the loading control γ-tubulin in cell lysates from four independent transfection experiments with 50 μg protein loaded per lane for the upper blot and 100 μg protein loaded per lane for the lower blot. To verify expression of HNF1α and HNF1β, membranes were stripped and re-probed with an antibody recognizing both HNF1α and HNF1β (upper blot) and with an antibody against HNF1β (lower blot). (C) ACE2 enzymatic activity was measured in extracts of 832/13 cells transfected with the HNF1α expression plasmid, the HNF1β expression plasmid, or the corresponding control plasmids. Each panel summarizes results for four independent transfection experiments. (D) Concentrations of ACE2 DPT and PPT were determined for βTC3 cells transfected with the HNF1α expression plasmid, the HNF1β expression plasmid, or the corresponding control plasmids. Samples giving no amplification were assigned a Ct value of 40 which is the highest cycle number in the assays. Each panel summarizes results for four independent transfections. **, ***: p < 0.01, p < 0.001 vs. control plasmid.

Article Snippet: An expression plasmid for human HNF1α (cat. no. SC300093) and a corresponding empty control plasmid pCVM6-XL5 were purchased from OriGene Technologies, Inc., Rockville, MD.

Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot, Activity Assay, Amplification

Journal: Biochimica et biophysica acta

Article Title: The transcription factor HNF1α induces expression of angiotensin-converting enzyme 2 (ACE2) in pancreatic islets from evolutionarily conserved promoter motifs

doi: 10.1016/j.bbagrm.2013.09.007

Figure Lengend Snippet: HNF1α induces ACE2 expression in pancreatic islet cells. (A) Concentrations of mRNA for RAS components and proteins involved in β-cell function relative to β-actin mRNA were determined for primary islet cells 24 and 48 h after being infected with the HNF1α-encoding adenovirus Ad-HNF1α or the control adenovirus Ad-GFP at MOI 100. AGT = angiotensinogen, PRR = (pro)renin receptor, ACE = angiotensin-converting enzyme, AT1a = angiotensin receptor type 1a, GLUT2 = glucose transporter 2, INS1 = insulin 1, INS2 = insulin 2. Concentrations are shown as the cycle-threshold (Ct) values for the target mRNA minus Ct for the normalizer β-actin mRNA obtained for RNA samples diluted to 0.5 ng/μl. Each data set was obtained from islet cells from 4-7 mice. *: p < 0.05 vs. Ad-GFP by pair-wise t-tests with p-values Bonferroni-adjusted by the number of comparisons. (B) For the mRNA targets that were significantly affected by infection with Ad-HNF1α, the Ad-HNF1α responses are calculated as the mRNA concentration for cells infected by Ad-HNF1α divided by the mRNA concentration for cells infected by Ad-GFP. (C) Insulin secretion at 2.8 and 25 mM glucose was determined for primary islet cells 41 h after being infected with Ad-HNF1α or Ad-GFP at MOI 100. Islet cells were from 3 mice, with each treatment combination of an adenovirus and a glucose concentration given to duplicate cell aliquots. (D) The concentration of ACE2 mRNA relative to β-actin mRNA was determined for primary islet cells 48h after being infected with Ad-HNF1α or Ad-GFP at MOI 100. Islet cells were from 3 mice infused with Ang II and 3 mice infused with saline. ***: p < 0.001 vs. Ad-GFP; #: p < 0.05 vs. saline. (E) Concentrations of mRNA for HNF1α, ACE2, and GLUT2 relative to β-actin mRNA were determined for primary islet cells 48 h after being infected with Ad-HNF1α or Ad-GFP at MOI 4 or 20. *: p < 0.05 vs. Ad-GFP by pair-wise t-tests with p-values Bonferroni-adjusted by the number of comparisons; @: p < 0.05 by pair-wise t-tests without adjustment for multiple comparisons. (F) Calculations of the Ad-HNF1α responses of HNF1α, ACE2, and GLUT2 for cells treated with adenovirus at MOI 4, 20, and 100.

Article Snippet: An expression plasmid for human HNF1α (cat. no. SC300093) and a corresponding empty control plasmid pCVM6-XL5 were purchased from OriGene Technologies, Inc., Rockville, MD.

Techniques: Expressing, Cell Function Assay, Infection, Concentration Assay

Journal: Biochimica et biophysica acta

Article Title: The transcription factor HNF1α induces expression of angiotensin-converting enzyme 2 (ACE2) in pancreatic islets from evolutionarily conserved promoter motifs

doi: 10.1016/j.bbagrm.2013.09.007

Figure Lengend Snippet: Comparison of the induction of ACE2 by HNF1α in islet cells and insulinoma cells. The mean concentrations of ACE2 mRNA and HNF1α mRNA relative to β-actin determined for islet cells infected for 48h with Ad-HNF1α or Ad-GFP and βTC3 cells transfected with the HNF1α expression plasmid or the control plasmid pCVM6-XL5 were compared. The calculations were based on the Ct values obtained in the quantitative RT-PCR experiments for Figs. 5 and 6.

Article Snippet: An expression plasmid for human HNF1α (cat. no. SC300093) and a corresponding empty control plasmid pCVM6-XL5 were purchased from OriGene Technologies, Inc., Rockville, MD.

Techniques: Infection, Transfection, Expressing, Plasmid Preparation, Quantitative RT-PCR