Transforming growth factor-beta induces airway smooth muscle hypertrophy

Abstract

Although smooth muscle hypertrophy is present in asthmatic airways, little is known about the biochemical pathways regulating airway smooth muscle protein synthesis, cell size, or accumulation of contractile apparatus proteins. We sought to develop a model of airway smooth muscle hypertrophy in primary cells using a physiologically relevant stimulus. We hypothesized that transforming growth factor (TGF)-beta induces hypertrophy in primary bronchial smooth muscle cells. Primary human bronchial smooth muscle cells isolated from unacceptable lung donor tissue were studied. Cells were seeded on uncoated plastic dishes at 50% confluence and TGF-beta was added. Experiments were performed in the absence of serum. TGF-beta increased cell size and total protein synthesis, expression of alpha-smooth muscle actin and smooth muscle myosin heavy chain, formation of actomyosin filaments, and cell shortening to acetylcholine. Further, TGF-beta increased airway smooth muscle alpha-actin synthesis in the presence of the transcriptional inhibitor actinomycin D, evidence that translational control is a physiologically important element of the observed hypertrophy. TGF-beta induced the phosphorylation of eukaryotic translation initiation factor-4E-binding protein, a signaling event specifically involved in translational control. Finally, two inhibitors of 4E-binding protein phosphorylation, the phosphoinositol 3-kinase inhibitor LY294002 and a phosphorylation site mutant of 4E-binding protein-1 that dominantly inhibits eukaryotic initiation factor-4E, each blocked TGF-beta-induced alpha-actin expression and cell enlargement. We conclude that TGF-beta induces hypertrophy of primary bronchial smooth muscle cells. Further, phosphorylation of 4E-binding protein is required for the observed hypertrophy.

Figure 1.

TGF-β increases airway smooth muscle cell size and protein synthesis. (A) Primary human bronchial smooth muscle cells cultured without or with TGF-β (1 ng/ml for 48 h). Left panels, phase contrast; right panels, FITC- phalloidin (magnification ×400). (B) Changes in primary human bronchial smooth muscle cell size after treatment with TGF-β (1 ng/ml for 72 h). Cells were sorted by forward scatter (left) and time of flight (right). TGF-β (1–10 ng/ml) increased cell size compared with 10% FBS and DMEM. The results shown are representative of three experiments. (C) Overall protein synthesis of human bronchial smooth muscle cells, with and without treatment with TGF-β (1 ng/nl for 24 h), as assessed by [³H]-leucine incorporation (n = 3, mean ± SEM).

Figure 2.

TGF-β alters airway smooth muscle contractile protein expression. (A) Representative immunoblots for α-smooth muscle actin, smMHC, and MLCK. TGF-β (1 ng/ml, 72 h) induced dose-dependent increases in α-smooth muscle actin expression. Total smMHC increased slightly, and there was shift in MLCK expression from the long to the short isoform. (B) Primary human bronchial smooth muscle cells were stained with Cy3-tagged anti–α-actin after treatment without (left panel, magnification ×400) or with TGF-β (1 ng/nl for 72 h, center and right panels, magnification ×400 and ×100, respectively). Most TGF-β–treated cells are thick with ample α-smooth muscle actin filaments. (C) Cells were stained with anti-smMHC plus FITC-labeled 2° antibody. After TGF-β treatment, a minority of cells contain smMHC filaments.

Figure 3.

TGF-β induces a contractile phenotype in primary bronchial smooth muscle cells. (A) Compared with untreated cells, TGF-β increases cell shortening in response to ACh (n = 50 for each group, mean ± SEM, *different from untreated cells, P < 0.05, ANOVA). (B) Phase contrast images of TGF-β–treated cells before (left) and after acetylcholine stimulation (right). Contracted cells round up in response to contractile agonists (magnification, ×100).

Figure 4.

Effects of TGF-β on α-smooth muscle actin steady-state mRNA level and stability. (A) Northern analysis of a-smooth muscle actin mRNA levels (TGF-β, 1 ng/ml for 48 h). (B) mRNA half-life was assessed by examining the level of α-smooth muscle actin mRNA after incubation with actinomycin D, a transcriptional inhibitor (5 mg/ml for 8–24 h). Group mean data are also shown (mean ± SEM).

Figure 5.

Effects of TGF-β on α-smooth muscle actin protein synthesis. Cells were treated with TGF-β (1 ng/ml) for 24 h and then incubated an additional 24 h with actinomycin D (5 mg/ml) and [³⁵S}methionine in the presence or absence of TGF-β. α-smooth muscle actin immunoprecipitates were resolved by SDS-PAGE and exposed to film. Group mean data (n = 3, mean ± SEM, *different from no TGF-β treatment, P < 0.001, ANOVA).

Figure 6.

Effects of chemical PI 3-kinase and mTOR inhibitors on 4E-BP phosphorylation, α-smooth muscle actin expression, cell proliferation, and protein synthesis. (A) Immunoblots showing effects of LY294002 (10 μM) and rapamycin (25 nM) on TGF-β–induced phosphorylation of 4E-BP1 and α-smooth muscle actin protein abundance. Group mean data for 4E-BP phosphorylation, as assessed by β + γ/α + β + γ ratio, are also shown (n = 3, mean ± SEM, different from TGF-β alone, *P < 0.001, **P = 0.005, ANOVA). (B) Effects of LY294002 (10 μM) and rapamycin (25 nM) on cell number (left), total protein synthesis (middle), and protein synthesis per cell (right). Left panel: n = 3, mean ± SEM, *different from control, P = 0.044; middle panel: n = 3, *different from control, P < 0.001, **different from TGF-β, P < 0.001; right panel: n = 3, *different from control, P < 0.001, **different from TGF-β, P < 0.001, ANOVA, ***different from TGF-β, P = 0.002. (C) Anti-p70 ribosomal S6 kinase immunoblot shows basal phosphorylation shift which is slightly increased by TGF-β treatment (1 ng/ml for 48 h). Pretreatment with rapamycin abolishes basal and stimulated phosphorylation shift, implying that mTOR is required for p70 ribosomal S6 kinase activation.

Figure 7.

Effects of chemical inhibitors (A) and mutant 4E-BP (B) on TGF-β–induced changes in cell size and α-smooth muscle actin expression, as assessed by Cy3-labeled anti–α-smooth muscle actin staining (magnification, ×200).