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* From University Children’s Hospital, Wuerzburg, Germany.
Correspondence to: Ulrike Bartram, MD, University Children’s Hospital, Josef-Schneider-Strasse 2, 97080 Wuerzburg, Germany; e-mail: bartram{at}mail.uni-wuerzburg.de
Abstract
Transforming growth factor (TGF) ß plays an important role in normal pulmonary morphogenesis and function and in the pathogenesis of lung disease. The effect of TGFß is regulated via a selective pathway of TGFß synthesis and signaling that involves activation of latent TGFß, specific TGFß receptors, and intracellular signaling via Smad molecules. All three isoforms of TGFß are expressed at high levels during normal lung development, being particularly important for branching morphogenesis and epithelial cell differentiation with maturation of surfactant synthesis. Small amounts of TGFß are still present in the adult lung, and TGFß is involved in normal tissue repair following lung injury. However, in a variety of forms of pulmonary pathology, the expression of TGFß is increased. These include chronic lung disease of prematurity as well as several forms of acute and chronic adult lung disease. While TGFß1 appears to be the predominant isoform involved, elevated levels of all three isoforms have been demonstrated. The increase in TGFß precedes abnormalities in lung function and detectable lung pathology, but correlates with the severity of the disease. TGFß plays a key role in mediating fibrotic tissue remodeling by increasing the production and decreasing the degradation of connective tissue via several mechanisms.
Key Words: lung disease • morphogenesis • pulmonary effects • pulmonary fibrosis • pulmonary surfactant • transforming growth factor ß
Transforming growth factor (TGF)ß is a member of a family of growth and differentiation factors with multiple functions in a variety of different organ systems. TGFß is notable for its capacity to modulate a variety of cellular behaviors, including cell proliferation, differentiation, and apoptosis.1 2 3 A comprehensive overview of the effects of TGFß has been given by Grande.1 TGFß isoforms have been shown to be essential for normal embryonic and fetal development and play a role in normal organ homeostasis and function.4 5 6 There also is increasing evidence for the importance of abnormalities of TGFß regulation or signaling in acute and chronic diseases.7 8 9 10 11 This review will focus on the role of TGFß during normal pulmonary morphogenesis and in the pathogenesis of lung disease.
Structural Characteristics of TGFß and Mechanisms of TGFß Signaling
TGFß exists in three isoforms (ß1, ß2, and ß3), which are structurally related, with a 60 to 80% sequence homology.1 The TGFß1, TGFß2, and TGFß3 genes have been mapped to chromosomes 19q13.1-q13.3,12 1q41,13 and 14q23–24,13 respectively. While many biological activities are identical or differ just in the intensity of the effect produced,1 some nonoverlapping functions have been discovered.
The pathway of TGFß signaling is summarized in Figure 1 . The TGFß genes are transcriptionally activated by interaction of the promoter region of the TGFß isoforms with distinct nuclear binding proteins.1 From TGFß messenger RNA, each isoform is initially synthesized as a larger precursor molecule containing the mature form of TGFß at the carboxyterminal region. This is proteolytically cleaved and secreted as an inactive homodimer that contains a latency-associated peptide. The latent TGFß is then activated extracellularly before receptor signaling.14 Activation can occur via transient acidification, alkanization, proteases (eg, plasmin or cathepsin),1 or substances that induce conformational rearrangement, such as thrombospondin-1.15 Potential mechanisms for TGFß regulation include transcriptional regulation of the TGFß genes, stability of TGFß messenger RNA, translation of messenger RNA, storage of TGFß, activation of latent TGFß, and inactivation of TGFß by circulating proteins and extracellular matrix macromolecules.1
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The intracellular signaling pathway downstream to the TGFß receptors is mediated by a family of transcription factors, the so-called Smad proteins.2 17 Activation of the type I receptor results in phosphorylation of the pathway-restricted Smad2 and Smad3, which then form a heteromeric complex with Smad4. The complex translocates to the nucleus where, either alone or in association with a DNA binding subunit, it activates target genes by binding to specific promoter elements.2 17 TGFß family members induce concentration-dependent responses that may be due to activation of promoters with different affinities for Smad-containing transcription factor complexes along a concentration gradient of TGFß.17 The regions of homology in the Smad molecules, termed MH1 and MH2, are important for modulating intracellular signaling. The MH1 domain plays a role as a negative regulator by interacting with the MH2 domain, thereby preventing heteromer formation between the Smad proteins.17 Smad6 and Smad7 function as inhibitors of TGFß signaling by binding to type I receptors and interfering with the phosphorylation Smad2 and Smad3.16 17 As expression of the inhibitory Smads is induced by TGFß, they may have a negative feedback role in signal transduction.
The Role of TGFß in Lung Development
The expression of TGFß during embryogenesis has been studied in detail in the mouse. All three TGFß genes are expressed at high levels during normal murine lung morphogenesis. TGFß1 is expressed as early as day 11 of gestation in the cytoplasm of the stromal and the epithelial cells of the primordial ducts that constitute the two major cell types of the developing lung. It appears to play a central role in lung branching and increases between day 14 and day 15 in the mouse, when differentiation of the primordial ducts into alveolar and bronchiolar ducts takes place.18 TGF-1 colocalizes with various extracellular matrix proteins, including collagen types I and III expressed at the epithelial-mesenchymal interfaces of stalks and clefts of the developing lung.18 TGF-2 transcripts have been found exclusively in the endodermal bronchiolar epithelium, the signal becoming stronger at later stages of development.19 TGF-3 is expressed from embryonic day 12.5 to day 15.5.19 Transcripts are first visible in the tracheal mesenchyme, followed by expression in the endodermal epithelial cells of the growing bronchioles in the mesodermal epithelial cells that give rise to the visceral pleura.19
At the level of the bronchi, human bronchial epithelial cells have been shown to have high-affinity receptors for TGFß, which is being the primary inducing factor for squamous differentiation.20 The influence of TGFß on the morphogenesis of the more distal parts of the lung appears to be exerted predominantly via effecting the developing type II cells as the stem cells of the complete alveolar epithelium and the only dividing cells with primary morphogenetic function in pulmonary acinus formation.21
Several studies have examined the effect of abnormal amounts of TGFß on lung development. Exogenous TGFß1 and TGFß2 inhibited branching morphogenesis in cultured mouse embryonic lung in a concentration-dependent manner.22 23 For TGFß1, this has been demonstrated to be associated with suppression of the proto-oncogene N-myc, which is expressed in epithelial cells involved in branching.22 Furthermore, in vivo and in vitro studies with overdoses of TGFß1 in mice resulted in disruption epithelial differentiation and inhibited the synthesis of Clara cell secretory protein, phospholipids, and the surfactant proteins A, B, and C.14 24 25
Knockout of the TGFß1 gene resulted in a diffuse inflammatory syndrome with pulmonary endothelialitis and interstitial pneumonia within 2 to 3 weeks after birth.4 6 However, abrogation of TGFß type II receptor signaling significantly stimulated lung branching morphogenesis in culture by preventing TGFß–induced epithelial cell G1 phase arrest and prevented TGFß1–induced down-regulation of epithelial differentiation marker genes such as surfactant protein C.23 26
Mice with targeted disruption of the TGFß2 gene died around birth from respiratory failure, but the lungs did not appear structurally abnormal.5 In contrast, TGFß3–null mutant mice had a specific neonatal lethal lung phenotype characterized by developmental delay with alveolar hypoplasia, lack of alveolar septal formation, and diminished expression of surfactant protein C.27
The intimate association between TGFß and lung development in humans is emphasized by the case of a neonate with pulmonary acinar aplasia. The lungs of this baby were extremely hypoplastic with complete absence of alveolar ducts and alveoli.28 In addition, the protein levels of TGFß1, TGFß2, and both the type I and the type II receptors were significantly lower than in normal controls. TGFß1 and type I receptor transcripts were reduced as well, whereas no differences were detected for TGFß3.28
Expression of TGFß in the Normal Lung
Small amounts of TGFß messenger RNA and protein as well as the type I and II receptors are still present in the normal lung after the completion of lung development. In mice, Coker et al29 found TGFß1 messenger RNA localized to bronchiolar epithelium, Clara cells, mesenchymal cells, vascular endothelium, and alveolar cells, including macrophages; in this study, TGFß3 messenger RNA was similarly distributed but not detected in the endothelium. In contrast, Pelton et al30 reported messenger RNA and protein of all three isoforms of TGFß to be expressed only in the proximal conducting airways, but not in alveoli of the distal airways. In their study,30 TGFß protein expression was confined to the bronchiolar epithelium while messenger RNA was found in smooth-muscle cells and connective tissue fibroblasts lying adjacent to the epithelium. Additionally, very high expression of all three TGFß messenger RNA transcripts was found in the smooth-muscle cells of the large vessels.30
In humans, bronchial epithelial cells contain the largest amounts of TGFß protein, the signal always being more intense at the apical pole of the cells.31 The TGFß isoforms and their receptors have also been demonstrated in alveolar macrophages,29 31 32 33 34 mesenchymal cells,29 vascular and airway smooth-muscle cells,31 32 33 34 and bronchial glands.33 Alveolar epithelial cells contain TGFß2 and TGFß3.34 While some studies29 32 33 also detected TGFß in normal epithelium, others34 reported epithelial presence of TGFß1 only in association with fibrosis.
The presence of TGFß in the normal lung suggests its participation in the normal regulation of physiologic processes to maintain lung homeostasis. These functions may include local immunomodulation, regulation of cell proliferation and differentiation, as well as the control of normal tissue repair.
The Role of TGFß in the Pathogenesis of Lung Disease
A common characteristic of many forms of lung disease is an inflammatory process with a phase of tissue injury followed by a phase of repair.9
35
Injury of lung tissue by chemical, bacteriologic, or immunologic noxious effects leads to an induction of TGFß that limits some of the inflammatory reactions and plays a key role in mediating tissue remodeling and repair.3
36
37
38
If the reparative processes are exaggerated and not adequately localized, lung pathology with fibrosis will ensue. This is typically associated with increased levels of TGFß and its receptors, and overexpression of TGFß has been shown to result in severe pulmonary fibrosis.39
In addition, several other cytokines, such as tumor necrosis factor (TNF)-
,40
keratinocyte growth factor,41
angiotensin II,42
and interleukin 1043
may exhibit profibrotic effects via the TGFß pathway.
An increased expression of TGFß messenger RNA8 32 35 44 45 46 and protein8 9 32 33 35 44 46 47 48 49 as well as the type II receptor32 has been documented during the phase of tissue remodeling in several forms of acute as well as chronic lung disease and systemic diseases involving the lung (Table 1 ).50 51 52 53 54 55 56 57 58 59 60 61 While most studies on human lung pathology focus on TGFß1, which appears to be the predominant isoform involved in pulmonary fibrosis, increases in all three isoforms have been demonstrated. This is consistent with findings in animals with bleomycin-induced lung fibrosis.62 63 64 65 66 67 68 69 70 71
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TGFß has also been implicated as playing a role in vascular remodeling in pulmonary hypertension, since increased levels of TGFß have been found in the lymph early during the development of induced pulmonary hypertension in sheep.74 In addition, TGFß1 increased the expression of fibronectin messenger RNA and protein as well as type IV collagen by pulmonary endothelial cells in vitro.75 However, Broekelmann et al44 did not find TGFß1 in the lung parenchyma of a patient with primary pulmonary hypertension.
Sources of TGFß in Lung Disease
Several cellular sources of TGFß appear to be activated during lung pathogenesis associated with fibrosis. The importance of these sources may vary at different stages of the reparative process and in different forms of lung disease.
Increased levels of TGFß have been demonstrated in epithelial cells and macrophages of the terminal airways and alveoli,32 33 35 45 48 53 54 56 60 and in subepithelial regions of dense fibroconnective tissue deposition.7 8 33 44 46 53 54 60 76 While during the early stages, platelets and epithelial cells may be a reservoir for TGFß,62 77 alveolar macrophages have been found to be important sources of increased production and secretion of TGFß during the phase with maximal TGFß levels in several forms of chronic lung disease.9 32 44 49 55 56 68 In neonates with respiratory distress syndrome, persistence of macrophages for > 4 days has been found to be predictive for the development of chronic lung disease.78
Similar results have been reported in an animal with bleomycin-induced pulmonary fibrosis, in which TGFß was found initially in the bronchial epithelium and the subepithelial matrix, while at the time of maximal TGFß expression it was present predominantly in macrophages in the alveolar interstitium.68
70
79
Mice with radiation-induced fibrosis exhibit increased TGFß expression by macrophages during the early phase of pulmonary injury, whereas later on type II pneumocytes and fibroblasts serve as important sources of TGFß.80
In patients with chronic obstructive lung disease, De Boer et al32
did not find an increase in TGFß expression by macrophages, but macrophages exhibited an increased expression of the TGFß type II receptor. In this study, the levels of TGFß also correlated with the number of mast cells.32
In patients with asthma, EG2-positive eosinophils represent the major source of TGFß1.46
TNF- appears to be important for this interleukin-5–mediated recruitment of eosinophils.81
Timing of TGFß Expression in Pulmonary Pathogenesis
The increase in TGFß is an early event in the process leading to chronic lung disease. It precedes abnormalities in lung function due to tissue remodeling and detectable fibrotic changes, but TGFß levels have been found to correlate with the severity of lung function abnormalities,32
46
lung pathology,46
47
and reduced survival times.82
The time course of TGFß expression during tissue remodeling has been studied in several animal models of pulmonary fibrosis. In rats, ventilation at extremely high pressures was associated with an increased expression of TGFß1 messenger RNA after 40 min.83
Induction of pulmonary fibrosis by intratracheal bleomycin instillation or thoracic irradiation in mice resulted in an increase of all three TGFß isoforms within hours after the injury.63
80
In a model with intraperitoneal bleomycin administration, causing a slower development of pulmonary fibrosis, TGFß1 messenger RNA was found to be decreased on day 1 followed by an increase.65
Depending on the type of injury, the levels of TGFß peaked around day 7 (intratracheal administration of bleomycin7
63
68
79
80
81
84
85
; overexpression of granulocyte macrophage colony-stimulating factor86
), day 14 (overexpression of TNF-39
) or 3 weeks (radiation-induced pulmonary fibrosis80
87
) in the areas developing fibrosis followed by a gradual decline. The up-regulation of TGFß always preceded the peak increase in extracellular matrix production.39
67
79
80
84
85
87
In humans, TGFß levels in the endotracheal aspirate of extremely low birth weight neonates are generally low in the first 24 h of life.50 51 Lecart et al51 found a subsequent increase in TGFß in these ventilated children with peak levels on days 20 to 25. Prenatal steroids significantly decreased the amount of TGFß.51 In lung tissue of premature babies who died following severe respiratory distress syndrome, Toti et al88 found macrophages immunoreactive for TGFß from days 4 to 16. High levels and an early rise of TGFß in the BAL fluid were predictive for the development of chronic lung disease of prematurity and the need for home oxygen therapy.50 51
In patients following lung transplantation, increases in TGFß have been found in the BAL fluid and in lung biopsy specimens before lung function became abnormal.9 45 However, in pulmonary diseases in which deterioration of lung function is primarily the result of increased bronchial obstruction, such as cystic fibrosis, early changes of lung function may be more characteristic for acute exacerbation while increased levels of TGFß1 are a sign of chronic progression of the disease with development of fibrosis.59
Limper et al8 found increased levels of TGFß1 messenger RNA in lung biopsy specimens to be characteristic for the early inflammatory lesions of patients with idiopathic pulmonary fibrosis, while TGFß1 protein was abundant in the fibroblast-rich, more advanced fibrotic pulmonary changes. In patients with coal worker’s pneumoconiosis and eosinophilic granuloma, increases in TGFß protein have been demonstrated in the early stages of the disease, whereas the advanced fibrotic lesions exhibited only weak expression of TGFß.49 55 Asakura et al58 reported that low expression of TGFß in old fibrotic lesions was associated with high levels of decorin, an extracellular matrix component. Since TGFß1 stimulates the synthesis of decorin and decorin in turn can bind TGFß, these data suggest that decorin may act as a negative feedback regulator of TGFß.
Profibrotic Effects of TGFß
Remodeling of lung tissue with deposition of connective tissue can be mediated by TGFß via several effects (Fig 2 ).
|
TGFß is the most potent direct stimulator of collagen production known, and all three isoforms have been demonstrated to increase collagen-expression in vitro.44 62 93 94 95 101 In addition, it induces the transcription and synthesis of various other components of the extracellular matrix by immature and mature pulmonary fibroblasts, such as fibronectin, glycosaminoglycans, and proteoglycans.1 44 95 101 102
In dermal fibroblasts, the chemotactic effects of TGFß1 have been shown to occur at concentrations much lower than those required for extracellular matrix induction.90 At higher concentrations, TGFß1 is no longer chemotactic. This makes it probable that TGFß1 attracts cells toward its source of delivery and highest concentration.98
Chemotaxis and Stimulation of Macrophages to Release Mesenchymal Growth Factors and Extracellular Matrix Proteins
TGFß is a chemoattractant for monocytes and macrophages.103
It also induces its own production by these attracted target cells,77
as well as the release of matrix glycoproteins such as fibronectin104
and various other mesenchymal growth factors including insulin-like growth factor-1,105
interleukin-1,67
106
107
alveolar macrophage-derived growth factor,104
109
TNF-,67
108
and PDGF.96
Interleukin-1 indirectly stimulates fibroblast proliferation by further inducing growth factors such as PDGF and interleukin-6.69 However, it may be only certain subsets of fibroblasts that respond to interleukin-1–mediated stimuli, since in mice a subset of pulmonary fibroblasts has been identified in which TGFß1 significantly down-regulates the expression of the interleukin-1 receptor type I, making these cells less responsive to interleukin-1–mediated induction of fibrosis.69
The isoforms of PDGF are known as potent chemoattractants and mitogens for fibroblasts, and at least part of the effect of TGFß on fibroblasts appears to be mediated via influencing the activity of PDGF.57
97
98
It has been suggested that PDGF and TGFß isoforms work in concert to stimulate the cellular events that result in fibrotic lesions, with PDGF being responsible for increased fibroblast proliferation during fibrogenesis and TGFß stimulating extracellular matrix production by fibroblasts.96
Data concerning the effect of TGFß on the PDGF receptors are inconclusive. While Bonner et al96
found a down-regulation of the PDGF- receptor subtype by TGFß in human lung fibroblasts in vitro,96
Ludwicka et al57
reported the opposite effect of TGFß on lung myofibroblasts of patients with scleroderma.
Stimulation of Extracellular Matrix and Growth Factor Synthesis by Type II Cells and Modulation of Type II Cell Differentiation and Function
In vitro studies110
111
112
have shown that TGFß1 can increase the synthesis of proteoglycans and fibronectin, as well as fibroblast growth factor-2 by alveolar type II cells. In addition, TGFß1 decreases messenger RNA expression of the surfactant proteins B and C by type II cells.112
113
The inhibition of surfactant protein B by TGFß1 occurs via blockage of protein kinase C-mediated nuclear translocation of the necessary transcription factors.113
Binding of fibronectin resulting in loss of type II cell characteristics and acquisition of a spread morphology and cytoskeletal structure resembling type I cells114
115
may add an indirect mechanism of inhibition of surfactant production by TGFß. Although the exact role of decreased surfactant proteins in TGFß–mediated structural pulmonary changes is at present not known, there is increasing evidence that dysfunction of the surfactant system contributes to the pathogenesis of both neonatal and acquired lung disease.116
Inhibition of Matrix Degradation
In addition to the promotion of matrix synthesis, TGFß stabilizes the newly formed extracellular matrix proteins by inhibiting their degradation. TGFß decreases the induction of the metalloproteinases collagenase and stromelysin that are induced by interleukin-1 and PDGF,117
118
while increasing the expression of protease inhibitors including the tissue inhibitor of metalloproteinase,117
and type-1 plasminogen activator inhibitor.119
120
121
These effects are achieved both by changes in gene transcription and changes in messenger RNA stability.1
Modulation of Cell-Matrix Interactions
TGFß regulates cell-matrix interaction by modifying the expression of cell-matrix adhesion protein complexes. TGFß–treated cells have an increased affinity for extracellular matrix components including fibronectin, collagen, and laminin. This effect is due to induction of integrin expression by TGFß.122
123
These interactions play an important role in tissue remodeling following injury.1
Genetic Polymorphism for TGFß
There is increasing evidence for a genetic predisposition to pulmonary disease associated with fibrosis due to increased synthesis of TGFß. Pretransplantation levels of TGFß were significantly higher in patients developing liver or lung fibrosis after autologous bone marrow transplantation than in those who did not.124
In the DNA sequence encoding the leader sequence of the TGFß1 protein, two genetic polymorphisms have been identified, located at codon 10 (either leucine or proline) and codon 25 (either arginine or proline).125 For both polymorphisms, the allele encoding proline has been shown to be associated with lower TGFß1 synthesis, and patients with the other alleles have been shown to be more prone to pulmonary fibrosis. Arkwright et al126 found the leucine allele at codon 10 to be associated with an accelerated decline in lung function in cystic fibrosis as compared with the TGFß1 low-producer phenotype on this codon. In this study, no difference was found between groups with regard to the polymorphism at codon 25.126 In patients undergoing lung transplantation for pulmonary fibrosis or cystic fibrosis, the proportion of cases with the TGFß1 high-producer genotype for both codon 10125 and codon 25125 127 was significantly increased, and lung allograft fibrosis occurred more frequently in patients that were homozygos for the codon 25 arginine/arginine allele.125 127
Potential Mechanisms for Influencing Pulmonary Tissue Remodeling Via Regulation of TGFß
Each of the steps along the pathway of synthesis, activation, and signaling of TGFß represents a potential mechanism for regulating the activity of TGFß (Fig 1) . While these mechanisms are primarily important for physiologic regulation of TGFß activity, there is increasing interest in using them to block excessive TGFß–mediated tissue response to fibrogenic stimuli.
A variety of substances have been shown to suppress the transcriptional up-regulation of TGFß in animal studies in vitro128
and in vivo.89
129
130
Knockout of the TNF receptor had the same effect,40
while keratinocyte growth factor, a potent growth factor for type II pneumocytes, prevented an increase in TGFß1 protein.42
In an in vitro study131
with human lung fibroblasts, interferon-
also exerted inhibitory effects on TGFß–induced fibrosis via activation of the signal transducer and activator of transcription STAT-1, which integrates the signals generated by TGFß. Interferon- has also been shown to be clinically effective in preventing deterioration of lung function and increasing the PO2 in patients with idiopathic pulmonary fibrosis.132
Mice lacking integrin vß6, which can bind and activate latent TGFß1, developed exaggerated inflammation, but were protected from pulmonary fibrosis.133
Several studies66 134 135 136 137 138 documented that prevention of receptor binding of TGFß on the target cell by neutralization of TGFß using TGFß1 or TGFß2 antibodies or a soluble version of the type II receptor significantly reduced bleomycin- or immune-induced pulmonary fibrosis in rodents in vitro and in vivo. Antibodies to TGFß1 also decreased fibrogenesis by human lung fibroblasts in vitro.139
In the mouse epithelial transfer of the decorin gene, a naturally occurring TGFß inhibitor that binds and neutralizes TGFß was also able to prevent TGFß–induced inhibition of normal lung development.140 In addition, TGFß-induced antiproliferative effects as well as the down-regulation of surfactant protein C were abrogated by decorin in cultured embryonic lungs, and lung branching inhibition by TGFß could be restored by the addition of decorin in culture.140
Smad7 suppressed bleomycin-induced pulmonary fibrosis in mice by blocking Smad2 phosphorylation, thus inhibiting intracellular transmission of the TGFß signal.141 Cyclosporin A inhibited TGFß–mediated stimulation of lung fibroblasts in vitro by direct inhibition of TGFß–induced activation of the transcription factor JunD.130 Interleukin 10 significantly reduced TGFß1–stimulated type I collagen expression in human pulmonary fibroblasts in vitro.43
In summary, TGFß plays an important role in normal lung morphogenesis and function as well as the pathogenesis of lung disease associated with fibrosis. TGFß is expressed at high levels during normal lung development, the expression of TGFß being particularly important for branching morphogenesis and epithelial cell differentiation with maturation of surfactant synthesis. TGFß is also involved in normal tissue repair following lung injury. Increased levels of TGFß resulting in increased production and decreased degradation of connective tissue play a key role in mediating fibrotic tissue remodeling in a variety of lung diseases.
Acknowledgements
We thank Matthias Emmert for assistance with the Figures.
Footnotes
Abbreviations: PDGF = platelet-derived growth factor; TGF = transforming growth factor; TNF = tumor necrosis factor
Received for publication May 8, 2003. Accepted for publication August 7, 2003.
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