Transforming Growth Factor-β 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)-␤ 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-␤ was added. Experiments were performed in the absence of serum. TGF-␤ increased cell size and total protein synthesis, expression of ␣-smooth muscle actin and smooth muscle myosin heavy chain, formation of actomyosin filaments, and cell shortening to acetylcholine. Further, TGF-␤ increased airway smooth muscle ␣-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-␤ 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-␤-induced ␣-actin expression and cell enlargement. We conclude that TGF-␤ induces hypertrophy of primary bronchial smooth muscle cells. Further, phosphorylation of 4E-binding protein is required for the observed hypertrophy.Keywords: 4E-binding protein; ␣-smooth muscle actin; eukaryotic initiation factor-4E; mammalian target of rapamycin (mTOR); phosphatidylinositol 3-kinase Increased airway smooth muscle mass is present in asthma. Ebina and coworkers (1) found two asthmatic subtypes, one with airway smooth muscle hypertrophy throughout the airways and another with hyperplasia in central bronchi. Benayoun and colleagues (2) found that patients with severe asthma had increased airway smooth muscle cell diameter and expression of ␣-smooth muscle actin and myosin light chain kinase (MLCK). On the other hand, Woodruff and coworkers (3) found that patients with mild asthma show no increase in cell size, though cell number was 2-fold higher. While smooth muscle mass increased by 50-83%, contractile protein mRNA expression was not changed, suggesting the importance of post-transcriptional mechanisms.(Received in original form May 2, 2005 and in final form October 4, 2005) These studies were supported by National Institutes of Health grants HL54685 and HL63314 (M.B.H), and DK42876 and DK057020 (K.N.B.).

Inhibition of Glycogen Synthase Kinase-3β Is Sufficient for Airway Smooth Muscle Hypertrophy

“…However, protein synthesis and ␣-actin expression induced by the pleotrophic growth factor TGF-␤ were not blocked by either GSK-3␤A9 or eIF2B siRNA, suggesting that, in the context of TGF-␤ treatment, an alternative pathway or pathways are sufficient for the hypertrophic response. As noted above, besides phosphorylation and inhibition of GSK-3␤, TGF-␤ treatment is also followed by phosphorylation of 4E-BP, which facilitates eIF4E-dependent translation of 7-methylguanine-capped mRNAs (27). Overexpression of AA-4E-BP, a nonphosphorylatable mutant, inhibited TGF-␤-induced airway smooth muscle hypertrophy.…”

“…More recently, cardiotrophin (CT)-1, a member of the IL-6 superfamily present in human lungs, has been shown to induce protein synthesis and cell enlargement, but not DNA synthesis, in cultured human bronchial smooth muscle cells (21) and guinea pig airway explants (22). In addition, we showed that transforming growth factor (TGF)-␤, a proasthmatic cytokine (23)(24)(25)(26), increased human bronchial smooth muscle cell size, protein synthesis, expression of ␣-smooth muscle actin and smooth muscle myosin heavy chain (smMHC), formation of actomyosin filaments, and cell shortening to acetylcholine (27). Further, TGF-␤ induced the phosphorylation of eIF-4E-binding protein (4E-BP), and inhibitors of 4E-BP phosphorylation blocked TGF-␤-induced ␣-actin expression and cell enlargement, suggesting that eIF4E-, cap-dependent translation is necessary for TGF-␤-induced hypertrophy.…”

Transforming Growth Factor β1 Function in Airway Remodeling and Hyperresponsiveness. The Missing Link?

“…TGF-b1 increases contractile protein expression in HASM, fibroblasts, and myofibroblasts. TGF-b1 induces the expression of SM22, calponin, a-SMA, MLCK, and Ca 21 pathway proteins in HASM cells, subsequently mediating enhancements in cell stiffness, actin-cytoskeleton reorganization, and contractility (87,104,105). In addition, mast cell-derived TGF-b1 may modulate the phenotype of HASM cells, promoting their differentiation into a more contractile phenotype and subsequently enhancing agonist-induced contraction (106).…”

Airway Smooth Muscle in Asthma Symptoms: Culprit but Maybe Innocent

“…Supports for this contention are accumulating. Cytokines such as interleukin (IL)-1 (11,92,200,250), tumor necrosis factor (TNF) (4,7,167,168,172,191,193,200,227), the combination of TNF and IL-1 (90,92), IL-13 (41,62,80,228), IL-5 (93, 250), IL-10 (79), granulocytemacrophage colony-stimulating factor (GM-CSF) (94), interferon (IFN) (5), leukemia inhibitory factor (LIF) (63,119) and transforming growth factor (TGF) (75,255), as well as the protease -tryptase (212,255) were shown to increase ASM contractility. In turn, the effect of these mediators is not always direct.…”

“…The molecular mechanisms governing the transformation of ASM into a hypercontractile phenotype may differ from one inflammatory mediator to another. In the case of TGF, increased expression of the contractile protein -SMA and actin filamentogenesis have been proposed (75,76). On the other hand, many inflammatory mediators increase ASM contractility through shared mechanisms related to alterations in calcium handling; either by Ca 2+ sensitization via the Rho-ROCK pathway or by increasing the intracellular mobilization of Ca 2+ via the CD38/cADPR/RyR pathway (both briefly described below and illustrated in Figure 4).…”