TGF-β2 reduces nitric oxide synthase mRNA through a ROCK-dependent pathway in airway epithelial cells

Abstract

Exhaled NO (eNO) is a potential noninvasive biomarker of inflammation in asthma. The significant intersubject variability of eNO within clinically similar patients has contributed to its limited clinical application. Arginase and NO synthase (NOS) utilize the same substrate (l-arginine) and contribute to the fibrotic and inflammatory features of asthma, respectively. Interestingly, TGF-β(2) can increase the expression of arginase, stimulates fibrosis, and is overexpressed in asthma. We hypothesized that TGF-β(2)-enhanced arginase activity would decrease gas phase NO release from lung epithelial cells by limiting l-arginine availability for NOS. Our results show that TGF-β(2) (5 ng/ml) significantly enhances total arginase activity up to two- to threefold in both primary small airway epithelial cells (SAECs) and the A549 cell line. Preincubation with TGF-β(2) prior to cytokine (IL-1β, TNF-α, and IFN-γ, 10 ng/ml each) stimulation decreases gas phase NO release to baseline levels (from 1.66 ± 0.52 to 0.30 ± 0.12 pl·s(-1)·cm(-2) and from 0.27 ± 0.03 pl·s(-1)·cm(-2) to near zero in SAEC and A549 cells, respectively). Addition of arginase inhibitor (N(ω)-hydroxy-nor-l-arginine) or small interfering RNA only partly reverses the reduction. In contrast, Rho-kinase (ROCK) pathway inhibitor (Y-27632) completely recovers the cytokine-induced NO flux in the present of TGF-β(2). Inducible NO synthase (iNOS) mRNA and protein levels change in a similar trend as NO release from the cells. We conclude that TGF-β(2) impacts cytokine-induced NO production in airway epithelial cells by reducing iNOS mRNA and protein levels through a ROCK-dependent pathway.

Fig. 1.

TGF-β₂ upregulates arginase activity and arginase I expression in lung epithelial cells. C, control; T, TGF-β₂; Cy, cytomix; Cy + T, cytomix + TGF-β₂. A: TGF-β₂ enhances arginase activity in small airway epithelial cells (SAECs) 24 h after stimulation. Preincubation and addition of TGF-β₂ to cytomix-treated SAECs increases total arginase activity throughout the experiment (n = 8–10). B: TGF-β₂ enhances arginase activity in A549 cells with the same trend as in SAECs (n = 8–10). C: representative Western blots and densitometry analysis (normalized by β-actin) indicates that TGF-β₂ increases arginase 1 (ARG1) protein expression in SAECs (n = 3). Spaces between lane T48 and lane Cy24 and between lane Cy + T24 and lane Cy48 indicate that the data are from 1 Western blot experiment, but not run in the same order as shown in the figure. D: quantitative PCR results demonstrate that TGF-β₂ increases arginase 1 mRNA expression in SAECs. Data are shown as means ± SD. *P < 0.05 compared with control group; ^P < 0.05 compared with cytomix group.

Fig. 2.

Preincubation of TGF-β₂ decreases gas phase NO release in cytomix-treated lung epithelial cells. Y, control + Y-27632; N, control + Nor-NOHA; Cy + T, cytomix + TGF-β₂; Cy + T + N, cytomix + TGF-β₂ + N^ω-hydroxy-nor-l-arginine (nor-NOHA); Cy + T + Y, cytomix + TGF-β₂ + Y-27632; Cy + Y, cytomix + Y-27632; T + Y, TGF-β₂ + Y-27632; Cy + T + siARG, cytomix + TGF-β₂ + small interfering RNA for arginase I. Cells were preincubated with TGF-β₂ for 24 h (t = 24 h) in Cy + T, Cy + T + N, and Cy + T + Y groups. At t = 0, fresh medium was supplied, and cytokines and inhibitors were added as the group names indicated. A: TGF-β₂ decreases cytomix-induced gas phase NO release from SAECs (n = 3). Nor-NOHA partly and Y-27632 completely reverses the reduction. B: total nitrate content in SAECs culture medium after 48-h exposure (n = 10). C: TGF-β₂ decreases cytomix-induced gas phase NO release from A549 cells. Nor-NOHA partly reverses the reduction, and Y-27632 significantly reverses the effect (n = 4). D: total nitrate content in A549 culture medium after 48 h exposure (n = 12, n = 3 for Cy + T + siARG). E: normalized inducible nitric oxide synthase (iNOS) mRNA expression in SAECs. TGF-β₂ decreases iNOS mRNA level, and addition of Y-27632 completely reverses the reduction in SAECs. F: representative Western blotting and densitometry analysis of iNOS protein expression normalized by β-actin in SAECs (n = 3). TGF-β₂ inhibits iNOS protein expression and Y-27632 completely reverses the inhibition. The space between control blot and the experimental conditions indicates that they were from 1 Western blot experiment, but not run in the same order as shown in the figure. Control blot is separately from treated conditions. Data are shown as means ± SD. *P < 0.05 compared with control group; **P < 0.01 compared with control group; #P < 0.05 compared with Cy + T group; &P < 0.05 compared with Cy + T + N group.

Fig. 3.

Intracellular l-arginine concentrations in small airway epithelial cells. The average concentration of intracellular l-arginine in SAECs is 2.68 mM. No significant difference was observed following different combinations of cytokine stimulation (n = 3). Data are shown as means ± SD.

Fig. 4.

Schematic for regulation of iNOS expression and NO release in airway epithelial cells. Cytomix stimulation of epithelial cells leads to an increase in iNOS mRNA increase at transcriptional level. TGF-β₂ decreases NO production by predominantly reducing iNOS mRNA expression or stability through a Rho-kinase (ROCK)-dependent pathway. TGF-β₂ enhanced arginase activity can reduce NO production, but to a small degree.