Figure 5.

Krüppel-like factor 2 (KLF2) promotes Rac1 activation and barrier function by transactivating endothelial Rap guanine nucleotide exchange factor 3 (RAPGEF3). (A) KLF2 overexpression restores Rac1 activation reduced by RAPGEF3 depletion. Human pulmonary microvascular cells (HMVEC) were transfected with 50 nM siRNA targeting RAPGEF3 (siRAPGEF3) or control siRNA (SC) for 24 hours followed by KLF2 overexpression via adenoviral transduction (ad-KLF2). Activated Rac1 pull-down assay was performed as previously described. The mean fold change of normalized activated Rac1 is shown on the right. n = 3. (B) KLF2 overexpression promotes monolayer integrity in HMVEC deficient for RAPGEF3, as shown by normalized TER. Quantification was conducted 48 hours after adenoviral transduction. n = 4. (C) KLF2 overexpression significantly increased RAPGEF3 mRNA expression in HMVEC transfected with siRAPGEF3 or SC. n = 8–11. (D) RAPGEF3 overexpression partially restores monolayer integrity in HMVEC deficient for KLF2 (left), as shown by normalized TER (right). HMVEC were transfected with 50 nM siKLF2 or SC, followed by RAPGEF3 overexpression via transfection with RAPGEF3 transcripts. Quantification was conducted 48 hours after RAPGEF3 overexpression. n = 4. All data are represented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, Student’s t test. ACTB = actin-β; ad-ctrl = adenovirus control; ctrl = control; Rac1 = Ras-related C3 botulinum toxin substrate 1; siKLF2 = siRNA against KLF2; siRNA = small interfering RNA; TER = transendothelial resistance.