Immunology and Cell Biology (2007) 85, 348–356 & 2007 Australasian Society for Immunology Inc. All rights reserved 0818-9641/07 $30.00 www.nature.com/icb
REVIEW
The regulatory role of TGF-b in airway remodeling in asthma Toluwalope Makinde1, Richard F Murphy1 and Devendra K Agrawal1,2,3 Both structural and inflammatory cells are capable of secreting transforming growth factor (TGF)-b and expressing TGF-b receptors. TGF-b can induce multiple cellular responses including differentiation, apoptosis, survival and proliferation, and has been implicated in the development of several pathogenic conditions including cancer and asthma. Elevated levels of TGF-b have been reported in the asthmatic airway. TGF-b binds to its receptor complex and activates multiple pathways involving proteins such as Sma and Mad homologues, phosphatidylinositol-3 kinase and the mitogen-activated protein kinases, leading to the transcription of several genes. Cell type, cellular condition, and microenvironment, all play a role in determining which pathway is activated, which, in turn, is an indication of which gene is to be transcribed. TGF-b has been shown to induce apoptosis in airway epithelial cells. A possible role for TGF-b in the regulation of epithelial cell adhesion properties has also been reported. Enhancement of goblet cell proliferation by TGF-b suggests a role in mucus hyper-secretion. Elevated levels of TGF-b correlate with subepithelial fibrosis. TGF-b induces proliferation of fibroblast cells and their differentiation into myofibroblasts and extracellular matrix (ECM) protein synthesis during the development of subepithelial fibrosis. TGF-b also induces proliferation and survival of and ECM secretion in airway smooth muscle cells (ASMCs), suggesting a possible cause of increased thickness of airway tissues. TGF-b also induces the production and release of vascular endothelial cell growth factor and plasminogen activator inhibitor, contributing to the vascular remodeling in the asthmatic airway. Blocking TGF-b activity inhibits epithelial shedding, mucus hyper-secretion, angiogenesis, ASMC hypertrophy and hyperplasia in an asthmatic mouse model. Reduction of TGF-b production and control of TGF-b effects would be beneficial in the development of therapeutic intervention for airway remodeling in chronic asthma. Immunology and Cell Biology (2007) 85, 348–356; doi:10.1038/sj.icb.7100044; published online 27 February 2007 Keywords: airway remodeling; chronic asthma; extracellular matrix; goblet cell hyperplasia; myofibroblasts; transforming growth factor-b
Airway remodeling is the modification of the normal structural properties of the airway wall and it entails changes in the composition and organization of its cellular and molecular constituents.1–4 Airway remodeling may be reversible or partially irreversible.1 The partially irreversible form of airway remodeling have been associated more with chronic asthma.2,5–7 Present asthmatic therapy with corticosteroids, theophylline and b2 agonists has been considerably successful in improving pulmonary airflow, but its effectiveness in reversing the structural remodeling in the airway of asthmatics has been limited.8–12 Treatment is most effective at the early stages of the development of asthma,13 but several asthmatic conditions still advance to the chronic form, which is responsible for mortality in both the young and old. Thus, novel therapeutic targets are still required so as to develop treatment and improve the quality of life for the cohort of patients that progress into the chronic stage of the disease and are nonresponsive to current treatment.
Airway remodeling plays an important role in the development of the symptoms associated with decreased pulmonary function in asthmatics.8,14,15 Airway remodeling occurs as an airway repair response to the injury sustained as a result of inflammation and disruption in this repair process leads to airway remodeling.8,15,16 Airway remodeling entails a complex array of events including epithelial layer damage, mucus gland and goblet cell hyperplasia, subepithelial fibrosis, airway smooth muscle cell (ASMC) hypertrophy and hyperplasia, and vascular remodeling.8,16–18 Each component of airway remodeling contributes to the overall pulmonary dysfunction.19 During the development of the asthmatic condition, infiltration of inflammatory cells occurs. Inflammatory cells as well as structural cells secrete cytokines, including transforming growth factor (TGF)-b, that regulate the airway remodeling process.20–25 TGF-b is particularly important, because it induces multiple effects in the same cell type or in different cells, depending on microenvironmental and
1Department of Biomedical Sciences, Creighton University School of Medicine, Omaha, NE, USA; 2Department of Internal Medicine, Creighton University School of Medicine, Omaha, NE, USA and 3Department of Medical Microbiology and Immunology, Creighton University School of Medicine, Omaha, NE, USA Correspondence: Professor DK Agrawal, Department of Biomedical Sciences, Internal Medicine, and Medical Microbiology and Immunology, Creighton University School of Medicine, CRISS II Room 510, 2500 California Plaza, Omaha, NE 68178, USA. E-mail:
[email protected] Received 22 August 2006; revised 29 November 2006; accepted 15 January 2007; published online 27 February 2007
TGF-b and airway remodeling in asthma T Makinde et al 349 Airway smooth muscle cell hyperplasia and hypertrophy
Vascular remodeling - VEGF production - PAI-1 production
- ASMC proliferation - ASMC survival
Goblet cell hyperplasia - Goblet cell proliferation
TGF-β
Epithelial layer damage - Epithelial cell apoptosis
- Increase mucus secretion
- Possible role in cell adhesion
Sub-epithelial fibrosis - Increase ECM production - Fibroblast differentiation to myo-fibroblast - Fibroblast and myo-fibroblast proliferation - Increase fibroblast survival
Figure 1 TGF-b induces various responses in different cells. These responses contribute to airway remodeling.
cellular conditions. Levels of TGF-b in the asthmatic airways are elevated.26,27 TGF-b also induces pulmonary fibrosis among other deleterious effects (Figure 1).22–24,28 Preformed and newly synthesized TGF-b can activate several pathways leading to gene transcription, by binding to TGF-b receptors on different cells that are involved in airway remodeling.29,30 The effects of TGF-b in relation to airway remodeling will be discussed in the following sections. EPITHELIAL LAYER DAMAGE In the asthmatic airways, the function of the epithelial layer is compromised. This is exemplified by increased epithelial shedding and damage to portions of the epithelial layer.8 A correlation between epithelial damage and airway hyper responsiveness has been suggested.31 The balance between proliferation and apoptosis is vital to the maintenance of the cell number and integrity in a particular location. The increase in loss of parts of the epithelial layer, as observed in the airway of asthmatics, may be owing to an imbalance between proliferation and apoptosis. Although proliferation of epithelial cells in the epithelial layer continues, the rate of damage may exceed the formation of new tissue. Simultaneously, decreased adhesion of the epithelial cells to the basement membrane could contribute to the epithelial layer shedding in the asthmatic airway.8 MUCUS GLAND AND GOBLET CELL HYPERPLASIA Increase in goblet cell proliferation and mucus hyper-secretion are congruent features of asthmatic airways. Mucus and goblet cells grow by branching out and elongating in response to the combined effects of inflammatory agents on the airway wall.8,17,32 As goblet cells are responsible for the production of mucus, their increased growth and stimulation results in an increase in mucus production and secretion.8,17,32 The mucus hypersecretion compliments the other airway wall modifications leading to occlusion of the airway and impairment in lung function.8,17,32 SUBEPITHELIAL FIBROSIS The subepithelial layer, also known as the lamina reticularis, is found under the subepithelial basal lamina. The sub-epithelial layer is composed of extracellular matrix (ECM) proteins including type 1, 3 and 5 collagen, proteoglycan, tenascin, and fibronectin.8,16,21,33 In
asthmatics, increased deposition of collagen, fibronectin and other ECM proteins in the sub-epithelial layer contribute to its swelling and stiffness.16 This condition is referred to as sub-epithelial fibrosis. In addition to its role in airway remodeling,34 subepithelial fibrosis has also been linked to airway hyper-responsiveness.31 In normal airways, collagen is distributed loosely in the ECM. In asthmatics, however, this loose distribution is replaced by a dense deposition of collagen network,8,16,30 which has been linked to airway distensibility, leading to impairment of lung function.16 It has been suggested that ECM proteins are secreted by subepithelial myofibroblasts because the number of myofibroblast cells correlates with the magnitude of subepithelial thickening.3,21 Myofibroblasts have a morphology intermediate between that of fibroblast cells and smooth muscle cells (SMCs). Myofibroblast cells are derived from fibroblasts and fibrocyte precursor.21 In the normal airways, deposition and degradation of ECM protein are in balance. For example, some cells secrete matrix metalloproteinase (MMP-9) that degrades collagen 4, a component of ECM in the basement membrane, and tissue inhibitor of matrix metalloproteinase (TIMP-1) that inhibits the degradative action of MMP-9.8,35–38 Imbalance between levels of MMPs and TIMPs in a particular location can lead to accumulation of ECM protein at that site.6,35,39 The lamina reticularis was an area of initial focus in research to investigate the changes in the composition of ECM. Other sites in the airways including adventitia are now gaining attention as potential sites for therapeutic intervention. The layer below the subepithelial layer is termed the bronchial layer. It consists of the inner airway wall, the airway smooth muscle layer, and the adventitia. The inner airway wall has a rich vascular network. The amount of elastic fiber in the inner wall of asthmatics appears to be increased. Abnormalities arise from fragmentation of elastin as well as increases and decreases in amounts of elastin at different levels of the airway.40–42 Some of the elastic fibers in the inner airway wall, which are attached to the subepithelial layer, are fragmented in the airway of asthmatics.41–43 All the above changes would impair the mechanical properties of the lungs. ASMC CELL HYPERTROPHY AND HYPERPLASIA Increase in airway smooth muscle layer thickness has been attributed to hyperplasia and hypertrophy of ASMCs.5,8,20,44,45 Apart from its functional contribution to airway remodeling, this increase in SMC Immunology and Cell Biology
TGF-b and airway remodeling in asthma T Makinde et al 350
layer thickness also functions in airway hyper-responsiveness.46 An increase in localized cell number could be a result of an increase in proliferation or an increase in survival rate or both.5,8,11,44 The increase in smooth muscle mass has also been attributed to several other factors. Increase in ECM between the ASMCs has been implicated in the ASMC bulk increase.5 Edema owing to plasma leakage, as a result of the abnormal properties in the blood vessels formed during angiogenesis in the asthmatic airway, is another factor contributing to ASMC mass.47 So also could be the micro-localization of mast cells in the ASMC tissue.48 Because of the increase in the smooth muscle cell hypertrophy and hyperplasia, the same degree of contraction of the cells would result in a greater overall force contributing to airway narrowing.49,50 MICROVASCULAR REMODELING Formation of blood vessels is abnormally increased in the asthmatic airway16,18,51,52 and this vascular remodeling contributes to airway narrowing. Blood vessels are formed in response to increased secretion of proangiogenic factors,16,18,51,53 which induce the proliferation of blood vessels. The blood vessels formed in the asthmatic airways have abnormal characteristics including increased permeability and upregulation of natural killer-1 receptors.18,54 An imbalance between vascular endothelial cell growth factor (VEGF) and angiopoietin-1 (Ang-1) has been implicated in the development of these abnormalities.47 Inflammatory cells and plasma extravasate because of increased permeability of newly formed blood vessels accumulate in nearby tissue leading to edema.16,18 TGF-b: STRUCTURAL CHARACTERISTICS, DISTRIBUTION, SOUCRES AND RECEPTORS TGF-b is a cytokine and a fibrogenic growth factor. It is a dimeric polypeptide with a 25 kDa molecular weight and can diffuse through basal compartments via cell junctions.23,55,56 Three isoforms of TGF-b are found in mammals.57,58 The role of TGF-b1 in airway remodeling and other pathologic conditions has been the most extensively investigated. It has been reported, however, that the level of TGF-b2 is greater than that of TGF-b1 in asthmatic and normal airways. The higher level of TGF-b2 in asthmatic airways is now regarded as distinguishing feature of the disease as levels of TGF-b1 do not change.59,60 TGF-b is primarily secreted by eosinophils, but other cells including macrophages, lymphocytes, fibroblasts, epithelial cells, and mast cells also secrete TGF-b.23,61,62 Other cytokines, including IL-5, IL-13 as well as TGF-b itself can induce the production and release of TGF-b from these cells.55,56,63–65 Cytokines such as interferon-g (IFN-g) can inhibit the production of TGF-b by targeting signaling involving Sma and Mad homologues (Smad).66 TGF-b is secreted in an inactive form, from which a latency associated peptide -1 must be removed to release the active peptide. This is catalyzed by several proteases including avb6 integrin, MMP-2 and -9, plasmin, thrombospodin-1, and calpains.23,56,67 TGF-b activation is tightly regulated since it can have various important consequences.56 TGF-b mediates its effects by activation of its receptors TGF-bR 1, 2, and 3, which have threonine/serine kinase intracellular domains that are phosphorylated on activation.56,58 TGF-b: SIGNAL TRANSDUCTION TGF-b can activate multiple intracellular pathways.23,55,63,68 Although the Smad proteins-mediated pathway is the primary pathway of TGFb signaling,69–72 TGF-b activates pathways mediated by other intracellular proteins depending on cell type and microenvironmental conditions. These mediating proteins include the phosphatidyl-inosiImmunology and Cell Biology
tol3 (PI3) kinase and mitogen-activated protein (MAPKS) kinases including extracellular signal-regulated kinase 12 (ERK1/2), TGF-bactivated kinase-1, p38 and c-Jun N-terminal kinase (JNK).56,68,73,74 The MAPKs function through a cascade of protein phosphorylations to elicit various cellular responses.55,56,74 When TGF-bR 1 and 3 are associated, TGF-bR1 binding to TGF-bR2 is enhanced.58 TGF-b forms a large complex made up of two TGF-b molecules and two molecules each of TGF-bR1 and TGF-bR2. This leads to subsequent phosphorylation of the TGF-bR1.68 Smad protein interacts with TGF-bRs through its highly conserved MAD-homology-2 domain. The Smad pathway can lead to stimulatory as well as inhibitory effects. TGF-b activates the Smad pathway through its TGF-bR1 by phosphorylation of the Smad 2/3 at the C-terminal end of the SXS motif on their MH-2 domains.56,61,68,71,72 The subsequent association of Smad 2, 3 and 4 forms a complex that translocates to the nucleus. The type of cofactors that they bind determines the transcription and activation of target genes56,75 such as those for ECM proteins genes and p21.76–78 The Smad 6 and 7 are involved in the inhibitory pathway that blocks the transcription of mRNA for the above proteins.8,16 Smad 7 acts as a negative feedback regulator by binding to TGF-bR1 and thereby preventing the phosphorylation of Smad 2/3 and further downstream signaling. Smad 7 expression is significantly lower in the asthmatic than the non-asthmatic airways. This is consistent with the increased fibrosis in asthmatic airways.8,16 The balance between the inhibitory and activating pathway is important in the determining the final outcome of TGF-b effect. TGF-b: PATHOPHYSIOLOGICAL ROLES IN AIRWAY REMODELING TGF-b plays several physiological and patho–physiological roles in the regulation of differentiation, apoptosis, survival and proliferation of various cells during embryonic development, wound healing, asthma and tumor growth. Elevated levels have been found in the airway of severe asthmatics. TGF-b has a half-life of 2 min in its free form and 90 min half-life in its latent form, so continuous accumulation of TGF-b in its latent form could eventually result in significant effects on various cell types (Figure 2).56 TGF-b IN EPITHELIAL LAYER DAMAGE Flow cytometric analysis has shown that an increased number of epithelial cells release TGF-b in asthmatics.44 Damage to the epithelial layer could be because of increased apoptosis of epithelial cells.12 Depending on the pathway activated, TGF-b can induce an antiapoptotic or an apoptotic effect in airway epithelial cells. An apoptotic effect will facilitate the development of an asthmatic phenotype characterized by increased epithelial damage, whereas an antiapoptotic effect would support epithelial cell hypertrophy. TGF-b elicits its antiapoptotic effect through the Smad 2/3 pathway. During overexpression of Smad7, the Smad 2/3 anti-apoptotic signaling is blocked and Smad 7 induces an apoptotic effect partly through the p38 MAPK pathway in airway epithelial cells.73,79–82 It has been reported that overexpression of Smad 2/3 leads to TGF-b-induced apoptosis in BEAS-2B airway epithelial cell line.73 TGF-b can also enhance the Fas-induced apoptotic effect in alveolar epithelial cells. Increased expression of TGF-b mRNA is associated with Fas-induced apoptosis and fibrosis in the alveolar epithelial cell. However, in central airway epithelial cells TGF-b induces an inhibitory effect on Fas-induced apoptosis.83 TNF-related apoptosis-inducing ligand (TRAIL) enhances TGF-b1 transcription, whereas TGF-b1 increases TRAIL expression in airway epithelial cells, suggesting interplay between TGF-b1 and TRAIL apoptotic signaling.27 In the asthmatic airway, the complex profile
TGF-b and airway remodeling in asthma T Makinde et al 351 EPITHELIAL CELLS
FIBROBLAST CELLS
AIRWAY SMOOTH MUSCLE CELLS
↑ VEGF TGF-β
ENDOTHELIAL CELLS
↑ VEGF TGF-β
Angiogenesis ↑ Proliferation ↑ Survival ↑ ECM production
Apoptosis
↑ Proliferation ↑ Survival
autocrine TGF-β
TGF-β
release
TGF-β
TGF-β
Survival ↑ Differentiation ↑ Survival ↑ ECM production
autocrine
autocrine ↑ ECM protein
TGF-β release
↑ VEGF
MYOFIBROBLAST
Figure 2 Several cells produce and secrete TGF-b. TGF-b diffuses between many tissues to elicit various functions of different cell types. Interplay of TGF-bmediated signaling between cells leads to exacerbation of airway remodeling.
of mediators may either upregulate or downregulate the various members of the Smad and MAPK pathways to induce a proapoptotic or antiapoptotic signal (Figure 3).73,83,84 It is evident that the epithelial layer is under stress because of continuous exposure to allergen and pro-inflammatory mediators. The p38 MAPK kinase-signaling pathway is activated in response to chemical agents or stress and it usually initiates survival and proliferation. However, in the presence of TGF-b, the p38 MAPK pathway is activated to initiate apoptosis.73 It has been suggested that, in the asthmatic airway, mediators either upregulate the p38 pathway or downregulate the Smad pathway, thereby supporting a TGF-b-induced apoptotic effect.12 Because of contextual dependence of TGF-b, reports of its apoptotic effects in epithelial cells and also the pathways that are activated conflict.56,67,73,79,81,82 There is consensus, however, that TGF-b induces apoptosis in airway epithelial cells and this may contribute to the airway epithelial layer damage in the asthmatic airway. TGF-b inhibits the expression of Muc4/SMC in epithelial cells of mammary gland and tumors. Muc4/SMC has antiadhesive, antirecognition and signal regulatory properties.85 It is involved in tumor metastasis;85 thus, the inhibitory effect of TGF-b on the expression of Muc4/SMC is lost in adenocarcinoma cells owing to defects in TGF-b signaling.85 This suggests a possible signaling role for TGF-b in epithelial layer shedding by regulating adhesive properties of the epithelial cells, leading to the detachment of the cells from the basement membrane. TGF-b IN MUCUS GLAND AND GOBLET CELL HYPERPLASIA Mucus-hyper secretion in the asthmatic airway could be caused either by increased secretion of mucus, increased proliferation of goblet cells or both.17,60 TGF-b2 levels correlate with mucin expression in the asthmatic airway. TGF-b2 induces mucin formation in bronchial epithelial cells by both increasing transcription and translation, although the mechanisms involved still need to be elucidated. Mucin is the major constituent of mucus in the airway. IL-13 induces expression of TGF-b2 more than of TGF-b1 in bronchial epithelial cells. It has also been reported that IL-13-induced mucus production is partly mediated by TGF-b2 upregulation. Treatment with antibody to TGF-b caused a reduction in the number of mucus-secreting goblet cells in an asthmatic mouse model.32,59,60 TGF-b increases IL-6
TGF-β1 P Pp TβR2 TβR1 P Smad 7
EPITHELIAL CELL
inhibition
inhibition
p38
Over-expression of cyclin D1 or E
P
Anti-apoptotic effect Inhibition of NF-kB Smad 2
Smad 3
P
P
+ Apoptotic effect
P
Smad 2
Smad 4
Smad 4
Smad 3 P
Cofactor binding determines binding location P
Smad 2 Smad 4 Smad 2 Smad 3 Co-activator P
p21
Figure 3 TGF-b induces apoptotic and antiapoptotic signals in airway epithelial cells. It contributes its antiapoptotic effect on Fas- or corticosteroid-induced apoptosis through the Smad2/3 pathway. The Smad 7 over-expression blocks the anti-apoptotic effect of the Smad2/3-mediated pathway. The antiapoptotic pathway is also blocked by overexpression of cyclin D1 or E. TGF-b induces its apoptotic signal through the Smads7 and p38 MAPK pathway.
expression in fibroblast cells and enhances mucus production and secretion. IL-6 can diffuse through the basement membrane to act on goblet cells.64 TGF-b IN SUBEPITHELIAL FIBROSIS ECM proteins are excessively deposited in subepithelial fibrosis.16,35 Mesenchymal cells are their primary source.22,57,62,68,75,86,87 TGF-b can directly or indirectly induce the proliferation fibroblast cells. At low doses, TGF-b induces the proliferation of fibroblasts and myofibroblast. It causes the release of fibroblast growth factor-2 (FGF-2).68,87 It has been reported that up-regulation of TGF-b production and release Immunology and Cell Biology
TGF-b and airway remodeling in asthma T Makinde et al 352 Same cell or CTGF
TGF-β
FGF-2
different cell p p Tβ
FGF-2 receptor
T R1
CTGF receptor
FGF-2release JNK dependent
p p
p JNK
P38 MAPK pathway
p
pathway
pathway
AP1 promoter
Cyclin mRNA
CTGF mRNA
Protein expression
Cyclin mRNA
Protein expression
DNA synthesis
MESENCHYMAL CELL
Cell proliferation Figure 4 TGF-b elicits cell proliferation on mesenchymal cells both directly and indirectly. The direct proliferative effect involves signaling through the p38 MAPK pathway. The indirect effect involves the expression of CTGF through a JNK-dependent pathway and the release of FGF-2. FGF-2 subsequently binds to its receptors on the same cell and elicits a proliferative effect through the JNK pathway. CTGF also binds to its receptor to signal a proliferative effect.
of connective tissue growth factor (CTGF) from fibroblast cells involves the PI3–JNK pathway,75 but ERK1/2 and Smad pathways have also been implicated.61 Both of the latter growth factors act in an autocrine manner to elicit a potent mitogenic effect (Figure 4). CTGF can enhance the synthesis of ECM proteins in mesenchymal cells in addition to their adhesion and migration.68,75,88 TGF-b can elicit an antiapoptotic effect in fibroblast cells.74,89 TGFb causes human bronchial fibroblast cells to undergo differentiation to a myofibroblast phenotype through the JNK pathway.39,86 The newly differentiated myofibroblasts have an increased capacity to express contractile proteins and deposit collagen.29,30,70 They constitutively express leukotriene C4 (LTC4) synthase and the Cys leukotriene-1 (CySLT1) receptor. CySLT1 receptor is a typical G-protein-coupled receptor with seven transmembrane regions and appears to conduct most of the signaling for Cyst-LTs.21,55,90 CysLTC4 is a proinflammatory lipid mediator that acts as a potent contractile agent in SMCS (Figure 5).21,65,90 It also increases the expression of TGF-b in epithelial cells.55 The newly expressed TGF-b can then diffuse through cell junctions to elicit effects on mesenchymal cells. This could, thus, be a form of epithelial–mesenchymal interaction. Leukotrienes can increase mucus production, mesenchymal proliferation, collagen synthesis, edema and epithelial cell proliferation.21,55 Fibroblast cells in different regions of the lung react differently in their procollagen synthesis and proliferative response to stimulation by TGF-b.29,91 Extent of fibrosis differ between compartments of the lungs. For example, abnormal loss of the ECM in the alveolar tissue of fatal asthmatics has been reported.40,43 Secretion of procollagen by proximal airway fibroblasts in response to TGF-b is greater than that by distal lung fibroblast, though distal lung fibroblast cells proliferate more rapidly and have more myofibroblast phenotypic changes.29 TGF-b can also induce collagen gel contraction through the Smad Immunology and Cell Biology
signaling pathway in lung fibroblast cells.92 It induces the expression of the a-SMA involved in SMC contraction.21,93 TGF-b induces the expression of MMPs and TIMPs, both of which are major counterbalancing regulators of ECM turnover.36,38,71,70 TGF-b can signal through the Smad7 pathway. Smad7 activation leads to the transcription of other ECM protein, decorin,16 which is a proteoglycan that is required in remodeling for crosslinking of the collagen fibrils. The amount of decorin determines how tightly or loosely collagen fibril associate and consequently how dense is the ECM. More tightly packed collagen bundles result in stiffer airway walls. Thus, tightness of the collagen bundle affects the elastic properties of the airway walls. Even in the presence of high amounts of collagen fibrils, if insufficient decorin is available, the collagen fibril is loosely packed. This results in decreased elastic recoil of the airway walls, leading to an impairment of lung function as seen in chronic asthma.16 TGF-b has been shown to induce mRNA transcripts of proteoglycan including biglycan, through the MKK6-p38 and Smad signaling pathways (Figure 6).33,57,76–78 TGF-b has a synergistic effect with major basic protein from eosinophils in significantly increasing the expression of IL-6.64,94 Upregulation of IL-6 expression in fibroblast cells that are stimulated with TGF-b has been reported and expression of IL-6 correlates with fibrosis. IL-6 has been linked with increased collagen synthesis, TIMP production and airway hyper-responsiveness.64,94,95 So, apart from its direct role, TGF-b contributes to fibrosis by inducing the expression of other mediators of fibrosis.93,94,96 TGF-b IN ASMC HYPERTROPHY AND HYPERPLASIA ASMCs can secrete all isoforms of TGF-b and also express TGF-bRs. TGF-b has been shown to increase the proliferation of ASMC through the MAPK pathway.47,97,98 TGF-b1 induces an increased synthesis of
TGF-b and airway remodeling in asthma T Makinde et al 353 Binds same cells or different cell TGF-β
LTC4
CysLT1 receptor
TβR complex Production of LTC4 synthase
p
-Proliferation of mesenchymal cells
p
P38 MAPK pathway
LTC4 synthase Activates the conjugation LTA4
-Broncho-constriction
+
Glutathione
-pro-inflammatory effect LTC4 AT F
p
Carrier-mediated export
TGF-β mRNA Figure 5 TGF-b induces synthesis of LT C4, which, in turn, induces the expression of TGF-b through the p38 MAPK pathway. LTC4 induces effects including proliferation, bronchoconstriction and inflammation.
TGF-β p p TβR2 TβR1
MESENCHYMAL CELLS
Smad 7
Blocks Pathway
MKK6 Smad 2
Smad 3
p
p
+ Smad 2
p p
p38
Smad 4
Smad 4
Smad 3
Cofactor binding determines binding location p
Smad 3
p
Biglycan mRNA
Decorin mRNA
Smad 4
Smad 2
Co-activator
c-Jun/c-Fos
Collagen mRNA
Figure 6 TGF-b induces the synthesis of mRNAs of various ECM proteins through different pathways.
ECM proteins and proliferation of ASMCs (Figure 6).44,97,99–102 TGFb can directly either induce the proliferation of ASMC or induce the production of CTGF through a combination of ERK and JNK pathways. CTGF is a potent mitogen for ASMCs and it can also induce the
expression of ECM proteins (Figure 4).88,97,103,104 TGF-b can also have a synergistic effect with other mediators, such as FGF-2.87 TGF-b induces the expression of VEGF in ASMC and VEGF modulates MMP9 synthesis.105 CTGF and VEGF may interact with the ECM to contribute to airway remodeling.30 TGF-b acts synergistically with fetal bovine serum to induce proliferation in ASMCs.44,97 During vascular remodeling in chronic asthma, in which TGF-b also plays a role, plasma leaks into the airway tissue including the ASMC layer.16,56,106 The plasma contains serum which, in the presence of TGF-b, would induce a significant proliferation of the ASMCs, thereby contributing to the ASMC hypertrophy and hyperplasia in airway remodeling. The proliferative effect of TGF-b is complex and context dependent. Depending on the dosage and microenvironment, TGF-b could either have a proliferative effect or inhibit proliferation of the same cells. At low dose and confluent conditions, TGF-b would induce the proliferation of ASMCs, whereas inhibiting growth of the same cells in subconfluent condition.44,97 The TGF-b effect is also time dependent as it as been shown that TGF-b inhibits proliferation of ASMCs seen after 24 h incubation and subsequently induces proliferation seen after 48–72 h.44,97 Some studies, however, have shown a continued proliferative process after 24 h.44 Variations in the experimental conditions and/or stimulatory components of conditioned media have been proposed as a possible explanation of the discrepancy. TGF-b can enhance survival of ASMC indirectly through ECM proteins or growth factor expression. It has also been reported that some ECM proteins interact with ASMC through integrins to induce an anti-apoptotic effect on ASMC (Figure 2).74,102,107–109 Since TGF-b induces the production of ECM proteins in ASMCs,99 this could constitute an indirect antiapoptotic effect of TGF-b on ASMCs. This increase in survival would contribute to the development of smooth muscle bulk. TGF-b also induces the expression of plasminogenactivator inhibitor (PAI-1) from ASMC. PAI-1 can induce migration Immunology and Cell Biology
TGF-b and airway remodeling in asthma T Makinde et al 354 TGF-β
Hypoxia
p p TβR2 TβR1
EPITHELIAL CELL
Smad 3
p
+ p
HIF-1α
Smad 3
Smad 4
Smad 4
Synergistic binding effect p
Smad 4 Smad 3
HIF-1α
Co-activator
VEGF mRNA Figure 7 TGF-b can induce VEGF mRNA synthesis through the Smad 2/3 pathway. TGF-b also acts synergistically with hypoxia-inducing factor (HIF)1a to increase the production of mRNA transcripts of VEGF.
of ASMCs and possibly contribute to the remodeling process.62,96,104 TGFb has also been shown to enhance ASMC responsiveness to bradykinin, a property that contributes to airway hyper-responsiveness.110 TGF-b has also been shown to have a synergistic effect with IL-1b and TNF-a in the expression of prostaglandins and Cox-1 in fibroblast cells. This further suggests a role for TGF-b in SMC contraction and the overall bronchoconstriction, as seen in the asthmatic airway.103 TGF-b IN MICROVASCULAR CONGESTION TGF-b is an angiogenic growth factor.111 However, it also paradoxically inhibits the growth of new blood vessels and thus could even have an apoptotic effect on endothelial cells.112 TGF-b can induce the production and secretion of several proangiogenic including VEGF and antiangiogenic growth factors through the Smad 3 pathway.16,18,30,56,95,106,113–115 Hypoxia augments the TGF-b-mediated production of VEGF (Figure 7).116,117 IFN-g inhibits the TGF-binduced expression of VEGF,115 which is a potent mitogen for endothelial cells. Elevated levels of VEGF in the asthmatic airways have been reported.16,54,106 This could also contribute to the imbalance between VEGF and Ang-1 resulting in abnormalities in the structural properties of the newly formed blood vessels in the asthmatic airway.18,53,54 TGF-b also induces the expression of PAI-1, which has a role in microvascular remodeling. The Smad 2/3 pathway is used to signal the expression of PAI-1.56,71,104 Apart from its role as a potent mitogen for endothelial cells, VEGF has also been shown to induce the production and release of MMP-9, suggesting another role for VEGF in pulmonary fibrosis.105 TGFb: THERAPEUTIC IMPLICATIONS It is evident that TGF-b plays a major role in the exacerbation of airway remodeling and several inhibitory agents have been reported.56 Immunology and Cell Biology
It has also been reported that several antiasthmatic therapies act partly through the inhibitory action of TGF-b. Theophylline inhibits TGF-b -induced procollagen secretion and differentiation of fibroblast cell into myofibroblast.118 Corticosteroids exhibit an inhibitory effect on the expression of TGF-b.55,119 However, in progressive fibrotic disease, corticosteroids do not inhibit the production of biologically active TGF-b production by alveolar epithelial cells, indicating a limitation to corticosteroid effectiveness in the inhibition of pulmonary fibrosis.23 Selective inhibitors such as SB-431542 have been shown to be effective in blocking phosphorylation of Smad3 during TGF-b signaling.120 Cytokines including IFN-g also inhibit TGF-b production.66 Therapeutic administration of antibody to TGF-b inhibits ECM deposition, ASMC proliferation, mucus production but does not affect established airway inflammation.60 Clearly, the interaction of TGF-b with its receptor is likely to remain a target in seeking opportunities for developing more successful therapeutic interventions. CONCLUSION TGF-b plays a multifunctional role in the development of airway remodeling. It induces apoptosis in airway epithelial cells and is possibly involved in the regulation of adhesion properties of epithelial cells leading to damage of the epithelial cell layer. TGF-b has a role in enhancing goblet cell proliferation and mucus secretion during mucus hyper-secretion. Some of the functions of TGF-b that contribute to subepithelial fibrosis include enhancement of fibroblast proliferation, differentiation and ECM protein production. TGF-b also enhances the proliferation of ASMCs and contributes in other ways to increase of the ASMC bulk. TGF-b is also involved in the abnormal proliferation of blood vessels leading to microvascular remodeling. All these properties of TGF-b confirm its importance in the exacerbation of airway remodeling. The studies reviewed also indicate that TGF-b does not have a set mode of action in the lungs. Its effects can change depending on context. How to manipulate the surrounding environment to achieve a desirable TGF-b effect should be explored. ACKNOWLEDGEMENTS This work was supported by a grant from the Nebraska Cancer and Smoking Related Diseases Program, Department of Health, Nebraska (to DKA), and by NIH grants R01HL070885 and R01HL073349 (both to DKA) and Carpenter Chair (to RFM) of Creighton University.
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