Transforming growth factor-beta modulates the expression of nitric oxide signaling enzymes in the injured developing lung and in vascular smooth muscle cells

Abstract

Nitric oxide signaling has an important role in regulating pulmonary development and function. Expression of soluble guanylate cyclase (sGC) and cGMP-dependent protein kinase I (PKGI), both critical mediators of nitric oxide (NO) signaling, is diminished in the injured newborn lung through unknown mechanisms. Recent studies suggest that excessive transforming growth factor-beta (TGF-beta) activity inhibits injured newborn lung development. To explore mechanisms that regulate pulmonary NO signaling, we tested whether TGF-beta decreases sGC and PKGI expression in the injured developing lung and pulmonary vascular smooth muscle cells (SMC). We found that chronic oxygen-induced lung injury decreased pulmonary sGCalpha(1) and PKGI immunoreactivity in mouse pups and that exposure to a TGF-beta-neutralizing antibody prevented this reduction of sGC and PKGI protein expression. In addition, TGF-beta(1) decreased expression of NO signaling enzymes in freshly isolated pulmonary microvascular SMC/myofibroblasts, suggesting that TGF-beta has a direct role in modulating NO signaling in the pup lung. Moreover, TGF-beta(1) decreased sGC and PKGI expression in pulmonary artery and aortic SMC from adult rats and mice, suggesting a general role for TGF-beta in modulating NO signaling in vascular SMC. Although other cytokines decrease sGC mRNA stability, TGF-beta did not modulate sGCalpha(1) or PKGIbeta mRNA turnover in vascular SMC. These studies indicate for the first time that TGF-beta decreases NO signaling enzyme expression in the injured developing lung and pulmonary vascular SMC. Moreover, they suggest that TGF-beta-neutralizing molecules might counteract the effects of injury on NO signaling in the newborn lung.

Fig. 1.

Transforming growth factor-β (TGF-β) neutralization increases soluble guanylate cyclase (sGC) and PKGI protein expression in the injured newborn lung. sGCα₁ and PKGI immunoreactivity were analyzed using distension-fixed lungs from mouse pups exposed to air or 85% O₂ and PBS or a TGF-β-neutralizing antibody (1D11), as indicated. A: sGCα₁ and PKGI immunoreactivity was observed in cells in walls of the alveoli and pulmonary arteries (open arrow) and veins (closed arrow) of the developing mouse pup lung. Laser scanning confocal micrographs of air-breathing mouse pup lungs are shown. Immunofluorescent (IF) control lungs were exposed to nonimmune IgG instead of the specific antibodies. Bar = 10 μm. B: sGCα₁ and PKGI immunoreactivity was increased in the hyperoxic lung exposed to TGF-β-neutralizing antibodies. Representative IF images of mouse pup lungs are shown. C: objective image analysis reveals that sGCα₁ and PKGI immunoreactivity levels in the peripheral lung are greatly decreased by 85% O₂, but expression is increased to nearly control levels with TGF-β neutralization. Violin plots of the immunoreactive signal luminosity are shown; abscissas represent the luminosity, and ordinates show the pixel frequency; white circles indicate the median values, and the black bars delineate the 1st and 3rd quartiles. D: percent fluorescent volume density (%FVD) of thresholded epifluorescent signals in the lung parenchyma. Data are means ± SD; n = 6 each group. *P < 0.05. E: immunoblotting also revealed that sGCα₁ and PKGI protein expression was improved in hyperoxic pup lungs exposed to TGF-β-neutralizing antibodies. Membranes with 60 μg of pup lung protein were probed with antibodies detecting sGCα₁ and PKGI to examine NO signaling enzyme protein expression and α-tubulin to confirm equal protein transfer.

Fig. 2.

Interstitial lung cells with smooth muscle cell (SMC) lineage isolated from the periphery of mouse pup lungs. A: a ∼1-mm-thick section of peripheral mouse pup lung tissue, containing terminal airway and microvascular structures as shown here in representative lung areas mapped by the arrows, was digested with collagenase, and cells were grown in the presence of serum on cell culture slides. B: nearly all cells isolated using this method exhibited α-smooth muscle cell actin (αSMA) and calponin immunoreactivity (as indicated; red). Cells exhibiting SMC/myofibroblast phenotype had an elongated shape with pseudopodia and strong αSMA immunoreactivity along prominent stress fibers; others exhibited less αSMA reactivity and organization, which is observed in pericytes. IF control cells were exposed to MOPC 21, a nonspecific monoclonal murine antibody (IgG₁). Bar = 10 μm. C: in contrast with adult mouse microvascular endothelial cells (PmvEC), the peripheral mouse pup lung SMC/myofibroblasts (PmvSMC) did not exhibit Griffonia simplicifolia IB4 lectin reactivity (green). Nuclei were identified with a DNA-binding fluorescent molecule (DAPI).

Fig. 3.

TGF-β₁ decreases sGCα₁ and PKGI immunoreactivity in peripheral mouse pup lung SMC. Shown are laser scanning confocal epifluorescent images of freshly isolated mouse pup lung PmvSMC treated with 10 ng/ml TGF-β₁ or vehicle for 6 h, fixed, and reacted with antibodies to sGCα₁ and PKGI and fluorescent secondary antibodies. IF control cells were exposed to preimmune serum instead of the specific antibodies; cell nuclei were detected with DAPI. Images are representative of 3 independent studies with similar results.

Fig. 4.

TGF-β modulates NO signaling enzyme mRNA levels in a rat pulmonary artery SMC (PASMC) line in a dose-dependent manner. Rat CS-54 PASMC were treated with the indicated levels of TGF-β₁ for 3 h, and NO signaling enzyme mRNA expression was examined using end-point RT-PCR. A: TGF-β₁ decreased sGC subunit and PKGIβ mRNA expression. Representative fluorescent images of ethidium bromide-stained amplicons from RT-PCR are displayed. B: densitometric analysis revealed that TGF-β₁ treatment decreased sGC subunit and PKGIβ mRNA levels. Target RNA levels were referenced to 18S RNA levels as described in materials and methods. Data are means ± SD, n = 4 per group. Data shown are representative of 3 independent experiments. *P < 0.05.

Fig. 5.

TGF-β modulates Smad2 phosphorylation and expression of NO signaling enzymes in CS-54 PASMC. Rat CS-54 PASMC were treated with 10 ng/ml TGF-β₁ for the indicated times. A: within 3 h of TGF-β₁ exposure, phospho- (p-) Smad2 levels increased in the CS-54 PASMC. Consistent total Smad2 and α-tubulin levels revealed that the TGF-β-mediated increase in p-Smad2 immunoreactivity was not due to differences in Smad2 expression or protein loading levels. The protein levels were determined using specific antibodies and immunoblotting. B: TGF-β₁ exposure decreased sGCα₁ and PKGIβ mRNA levels (compared with 18S) in CS-54 PASMC as detected by end-point RT-PCR. Data are means ± SD, n = 4 per group. Results shown are representative of 3 independent experiments. *P < 0.05.

Fig. 6.

TGF-β stimulates Smad-dependent gene expression and decreases sGC and PKGI mRNA expression in murine vascular SMC. The indicated mouse vascular SMC were treated with 10 ng/ml TGF-β₁, and the levels of mRNA encoding NO signaling enzymes were determined using end-point RT-PCR. TGF-β decreased sGCα₁, sGCβ₁, and PKGIβ mRNA levels (compared with 18S) in murine SMC from PASMC or aorta (AoSMC) within 3 h of exposure. Cells were exposed to 10 ng/ml TGF-β₁ for the indicated times. Data are means ± SD; n = 4 per group. Results shown are representative of 3 independent experiments. *P < 0.05.

Fig. 7.

Cell density modulates the regulation of PKGIβ mRNA levels by TGF-β. Early passage rat AoSMC were seeded on cell culture plates at indicated densities. After 48 h of culture, cells were treated without or with 10 ng/ml TGF-β₁ (open and closed boxes, respectively) for 6 h. PKGIβ and sGCα₁ mRNA levels, relative to 18S RNA levels, were determined using end-point RT-PCR. Although the decrease in sGCα₁ mRNA levels caused by TGF-β₁ treatment was not affected by cell density, the effect of TGF-β₁ on PKGIβ mRNA levels was seen only at higher SMC densities. Data are means ± SD, indexed to the relative mRNA levels obtained in the high-density cells without TGF-β treatment, and typical of 3 independent experiments; n = 6 per group; *P < 0.05.

Fig. 8.

TGF-β does not regulate stability of sGCα₁ and PKGIβ mRNAs. Rat AoSMC were treated as indicated with or without 10 ng/ml TGF-β₁ and 10 μM actinomycin D (Act D) for 6 h. sGCα₁ and PKGIβ mRNA levels were determined using end-point RT-PCR. Results are relative to the mean mRNA values observed in the cells treated with media containing vehicle (DMSO) alone. There was no significant difference between cells treated with media with or without DMSO (data not shown). The decrease in sGCα₁ and PKGIβ mRNA levels observed in the SMC treated with Act D was not further exacerbated with TGF-β₁ treatment. Data are means ± SD and typical of 3 independent experiments; n = 9 per group; *P < 0.05 vs. other treatments.