Mechanical stress induces pre-B-cell colony-enhancing factor/NAMPT expression via epigenetic regulation by miR-374a and miR-568 in human lung endothelium

Abstract

Increased lung vascular permeability and alveolar edema are cardinal features of inflammatory conditions such as acute respiratory distress syndrome (ARDS) and ventilator-induced lung injury (VILI). We previously demonstrated that pre-B-cell colony-enhancing factor (PBEF)/NAMPT, the proinflammatory cytokine encoded by NAMPT, participates in ARDS and VILI inflammatory syndromes. The present study evaluated posttranscriptional regulation of PBEF/NAMPT gene expression in human lung endothelium via 3'-untranslated region (UTR) microRNA (miRNA) binding. In silico analysis identified hsa-miR-374a and hsa-miR-568 as potential miRNA candidates. Increased PBEF/NAMPT transcription (by RT-PCR) and expression (by Western blotting) induced by 18% cyclic stretch (CS) (2 h: 3.4 ± 0.06 mRNA fold increase (FI); 10 h: 1.5 ± 0.06 protein FI) and by LPS (4 h: 3.8 ± 0.2 mRNA FI; 48 h: 2.6 ± 0.2 protein FI) were significantly attenuated by transfection with mimics of hsa-miR-374a or hsa-miR-568 (40-60% reductions each). LPS and 18% CS increased the activity of a PBEF/NAMPT 3'-UTR luciferase reporter (2.4-3.25 FI) with induction reduced by mimics of each miRNA (44-60% reduction). Specific miRNA inhibitors (antagomirs) for each PBEF/NAMPT miRNA significantly increased the endogenous PBEF/NAMPT mRNA (1.4-3.4 ± 0.1 FI) and protein levels (1.2-1.4 ± 0.1 FI) and 3'-UTR luciferase activity (1.4-1.7 ± 0.1 FI) compared with negative antagomir controls. Collectively, these data demonstrate that increased PBEF/NAMPT expression induced by bioactive agonists (i.e., excessive mechanical stress, LPS) involves epigenetic regulation with hsa-miR-374a and hsa-miR-568, representing novel therapeutic strategies to reduce inflammatory lung injury.

Figure 1.

Effects of inflammatory agonists on pre–B-cell colony-enhancing factor (PBEF)/NAMPT expression and 3′-untranslated region (UTR) reporter activity. Total RNA was isolated from human pulmonary artery endothelial cells (ECs) challenged with control vehicle or LPS (100 ng/ml) or exposed to 18% cyclic stretch (CS) for 0 to 8 hours. PBEF/NAMPT mRNA levels were detected via real-time PCR (A). Data are presented as fold change in mRNA level over vehicle-treated control and expressed as mean ± SE from three independent experiments. *P < 0.05 versus unstimulated control. ^#P < 0.01 versus unstimulated control. Confluent ECs were treated with control vehicle or 18% CS (B) and LPS for the indicated times, and endogenous PBEF was detected via immunoblot. The bar graphs represent relative densitometry (18% CS [C]; LPS [D]). Data are presented as fold changes in PBEF/NAMPT over vehicle-treated control and expressed as means ± SE from three independent experiments. *P < 0.05 versus unstimulated control. ^#P < 0.01 versus unstimulated control. ECs were cotransfected with PBEF/NAMPT 3′-UTR reporter together with phRL-TK, a Renilla luciferase normalization control vector, and exposed to 18% CS (E) or treated with LPS (F) (24 h), and luciferase activity was measured using the Dual Luciferase Assay System according the manufacturer’s protocol. TNF-α luciferase reporter was used as a positive control. The bar graph represents relative luciferase units. Data are presented as relative luciferase units over vehicle-treated control and expressed as means ± SE from three independent experiments. *P < 0.05 versus unstimulated control. ^#P < 0.01 versus unstimulated control.

Figure 2.

Time-dependent effects of inflammatory agonists on mature miR-568 expression in human lung ECs. Total RNA was isolated from ECs and treated with control vehicle, 18% CS, or LPS (100 ng/ml) for 0 to 24 hours, and the level of miR-568 was determined via real-time PCR. Data are presented as fold change in microRNA level over vehicle-treated control and expressed as means ± SE from three independent experiments. *P < 0.05 versus unstimulated control. ^#P < 0.01 versus unstimulated control.

Figure 3.

Effect of miR-568 mimic on LPS-induced human lung EC dysfunction. ECs grown in chambers on gold microelectrodes were transfected with miR-568 mimic (A) or treated with nonspecific negative control mimic (nc) as described in Materials and Methods and used for transendothelial electrical resistance (TER) measurements. At time = 0, cells were stimulated with LPS (100 ng/ml) or vehicle control. Shown are pooled TER data of five independent experiments. (B) Maximal value of normalized TER in RCs with nc reagent (control) achieved within 12 hours was taken as 100% ± SE. *Significantly different from cells treated with nc reagent with LPS (P < 0.01).

Figure 4.

Effect of miRNA mimics on inflammatory agonist–induced PBEF/NAMPT transcription in human lung ECs. Total RNA was isolated from ECs, transfected with nc or with the indicated miRNA mimics (48 h) and untreated (A and B), exposed to 18% CS (A, 2 h), or treated with LPS (B, 4 h). PBEF/NAMPT mRNA levels were detected via real-time PCR. Transfection was performed with miR-1290 used as the second negative control. Data are presented as fold change in mRNA level over vehicle-treated control and expressed as means ± SE from four independent experiments.

Figure 5.

Effect of microRNA (miRNA) mimics on inflammatory agonist–induced PBEF/NAMPT expression in human lung ECs. ECs were transfected with nc or with the indicated miRNA mimics (48 h) and exposed to 18% CS (10 h) or LPS (B, 48 h), and endogenous PBEF/NAMPT protein was detected via immunoblots. The bar graphs represent relative densitometry (18% CS [A]; LPS [C]). Data are presented as fold changes in PBEF/NAMPT compared with nc or corresponding miRNA pretreatment without agonist challenge and expressed as means ± SE from three independent experiments. *P < 0.05 versus unstimulated control. ^#P < 0.01 versus unstimulated control.

Figure 6.

Effect of miRNA mimics on inflammatory agonist–induced PBEF/NAMPT 3′-UTR reporter activity in human lung ECs. ECs were cotransfected with PBEF/NAMPT 3′-UTR reporter together with nc or with the indicated miRNA mimics and phRL-TK, a Renilla luciferase normalization control vector. ECs were untreated and exposed to 18% CS (A) or treated with LPS (C) (24 h) (B), and luciferase activity was measured according the manufacturer’s protocol. Data are presented as relative luciferase units over vehicle-treated control and expressed as means ± SE from four independent experiments. *Significantly different from control cells without stimulation (P < 0.05). **Significantly different from control cells stimulated with 18% CS or LPS (P < 0.05). ^#Significantly different from control cells stimulated with 18% CS or LPS (P < 0.01).

Figure 7.

Effects of miRNA antagomirs on inflammatory agonist–induced PBEF/NAMPT expression and 3′-UTR reporter activity. Total RNA was isolated from ECs and transfected with nc or with the indicated miRNA. Anti- and PBEF/NAMPT mRNA levels were detected via real-time PCR (A). Data are presented as fold change in mRNA level over control and expressed as means ± SE from four independent experiments. *Significantly different from control cells (P < 0.05). ^#Significantly different from control cells (P < 0.01). ECs transfected with nc or with the indicated miRNA anti- and endogenous PBEF/NAMPT protein were detected via immunoblot. The bar graph represents relative densitometry (B). Data are presented as fold changes in endogenous PBEF/NAMPT over nc pretreatment and expressed as means ± SE from three independent experiments. *P < 0.05 versus control. ECs were cotransfected with the PBEF/NAMPT 3′-UTR reporter together with phRL-TK, nc, or with the indicated miRNA (C), and luciferase activity was measured according the manufacturer’s protocol. Data are presented as relative luciferase units over control and expressed as means ± SE from four independent experiments. *Significantly different from control cells (P < 0.05).