Figure 4.

Krüppel-like factor 2 (KLF2) regulates pulmonary microvascular Rap guanine nucleotide exchange factor 3 (RAPGEF3) expression through RAPGEF3 promoter activity. In human pulmonary microvascular cells (HMVEC) deficient for KLF2 (siKLF2), (A) RAPGEF3 mRNA and (B) RAPGEF3 protein expression are significantly reduced. In HMVEC overexpressing KLF2 via adenoviral transduction (ad-KLF2) (C) RAPGEF3 mRNA, and (D) RAPGEF3 protein expression are elevated compared with control cells (ad-ctrl). n = 5–10. (E) In HMVEC overexpressing KLF2 via transfection with KLF2 transcripts (T7-KLF2), RAPGEF3 mRNA expression is significantly increased compared with cells transfected with AUG-mutated control transcripts (T7-ctrl). n = 7. (F) Schematic representation of RAPGEF3 promoter containing multiple CACCC motifs (WT) and mutated RAPGEF3 promoter with five CACCC motifs replaced by GTACT (mut). (G) Intrinsic promoter activity of RAPGEF3 promoter reporter. HEK cells were cotransfected with RAPGEF3 promoter constructs (WT or mut) or the promoter-less vector (basic) and pRL-TK. Promoter activities were indicated by the dual luciferase ratio. (H) KLF2 overexpression via plasmid transfection promotes RAPGEF3 promoter activity, and enhancement of KLF2 on RAPGEF3 promoter activity was diminished in mutated RAPGEF3 promoter. n = 4–13. (I) ChIP–quantitative polymerase chain reaction analysis from HMVEC transfected with KLF2 transcripts with HA tag. ChIP was performed with either a control IgG antibody or the antibody against HA followed by quantitative polymerase chain reaction using primers for RAPGEF3 promoter. n = 5. All data are represented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, Student’s t test. ACTB = actin-β; ChIP = chromatin immunoprecipitation; HA = hemagglutinin; HEK = human embryonic kidney; pRL-TK = Renilla luciferase control reporter vectors; SC = control siRNA; siRNA = small interfering RNA; WT = wild type.