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Respiratory Research Group, Department of Pharmacology, and Discipline of Medicine, The University of Sydney
Woolcock Institute for Medical Research, Royal Prince Alfred Hospital
Department of Endocrinology, Royal Prince Alfred Hospital, Sydney, Australia
ABSTRACT
Transforming growth factor (TGF)- and connective tissue growth factor may be implicated in extracellular matrix protein deposition in asthma. We have recently reported that TGF- increased connective tissue growth factor expression in airway smooth muscle cells isolated from patients with asthma. In this study, we examined fibronectin and collagen production and signal transduction pathways after stimulation with TGF- and connective tissue growth factor. In both asthmatic and nonasthmatic airway smooth muscle cells, TGF- and connective tissue growth factor led to the production of fibronectin and collagen I. Fibronectin and collagen expression was extracellular regulated kinase–dependent in both cell types but phosphoinositide-3 kinase–dependent only in asthmatic airway smooth muscle cells. p38 was implicated in fibronectin but not collagen expression in both cell types. TGF- induction of fibronectin and collagen was in part mediated by an autocrine action of connective tissue growth factor. Phosphorylation of SMAD-2 may represent an additional pathway because this was increased in asthmatic cells. Our results suggest that these two cytokines may be important in the deposition of extracellular matrix proteins and that the signal transduction pathways may be different in asthmatic and nonasthmatic cells.
Key Words: asthma connective tissue growth factor extracellular matrix transforming growth factor
Airway remodeling is one of the hallmark features of asthma, and changes in the deposition of extracellular matrix (ECM) proteins are likely to constitute a contributing factor.
Histologic studies of airways from patients with and without asthma describe enhanced deposition of collagen I, III, and V; fibronectin, tenascin; hyaluronan; versican; laminin 2/2; lumican; and biglycan (1–4), whereas levels of collagen IV and elastin are decreased (5). The ECM is produced by a variety of cells, including those from the fibroblast/myofibroblast lineage and airway smooth muscle (ASM) cells.
Only a few histologic studies have examined the changes in ECM proteins specifically in the area of the ASM in asthmatic airways. Bai and colleagues observed an increase in the amount of total ECM around individual airway smooth muscle cells in fatal asthma cases (6). Others have reported an increase in collagen (7), hyaluronan, and versican (1). Moreover, we have observed that the profile of ECM proteins produced in vitro from asthmatic ASM differs from nonasthmatic cells (8) in that production of perlecan and collagen I by asthmatic ASM cells were significantly increased, whereas laminin 1, chondroitin sulfate, and collagen IV were decreased.
The mechanisms leading to the deposition of ECM proteins in the airways of patients with asthma are unknown; however, in other fibrotic diseases, both transforming growth factor (TGF-) and connective tissue growth factor (CTGF) have been implicated (9). TGF- is a 25-kD protein that is secreted as an inactive product noncovalently bound to a latency-associated protein. Release of active TGF- occurs through the action of proteolytic enzymes such as plasmin or cathepsin. TGF- is produced by many cells, including ASM. It is a cytokine that stimulates ASM cells to produce a variety of ECM proteins (10, 11). In bronchoalveolar lavage fluid from patients with asthma, levels of TGF- protein are increased (12) and gene expression is increased in the bronchi (13). Furthermore, in the asthmatic airway mucosa, the levels of TGF- are increased and correlate with the thickness of the reticular basement membrane, supporting a role for TGF- in remodeling in asthma (14).
The mechanisms of TGF- signaling are not completely understood. The TGF- receptor on the cell surface is a heterotetrameric complex consisting of two transmembrane receptor serine/threonine kinases with a type II ligand binding receptor (TGF--RII) and a type I signaling receptor (TGF--RI). The classic signaling cascade after TGF- binding to this receptor complex is via the Smad pathway. Smads 2 and 3 are direct substrates of TGF--RI and, together with Smad 4, play important roles as cytoplasmic signaling molecules. Although the Smad pathway has been the focus of work characterizing TGF- signaling, it has recently been shown that the TGF--RI/II complex can also signal through other pathways, including the mitogen-activated kinases and phosphoinositol-3 kinase (PI3K) (15, 16).
Many of the TGF-–induced fibrotic effects are mediated by CTGF. CTGF is an immediate early gene of the CCN family that was first described as a growth factor from human umbilical vein endothelial cells (17). It is a 38-kD, cysteine-rich protein that has been shown in human lung fibroblasts to upregulate gene expression and protein production of both collagen I and fibronectin (18). We have previously shown in ASM cells that TGF- upregulates both CTGF mRNA and protein, an effect that was significantly greater in cells obtained from patients with asthma (19). Thus, this pathway may be pivotal in the ECM deposition that characterizes remodeling.
The aims of this study were to define the pathways linking TGF-, CTGF, and production of fibronectin and collagen in asthmatic and nonasthmatic ASM cells.
Some of the results from this study have previously been presented in abstract form (20).
METHODS
Chemicals
Details about sources of all chemicals and reagents are provided in the online supplement.
RhCTGF protein was produced using the AdEasy recombinant human adenoviral expression system, as described previously (21, 22); full details are provided in the online supplement.
Cell Culture
Human ASM cells were obtained from patients without (59 ± 17 yr; mean ± SD) and with asthma (37 ± 16 yr; mean ± SD) by methods adapted from those previously described (23, 24). The characteristics of the patients are listed in Table 1. Approval for all experiments using human lung was provided by the Ethics Committee of the University of Sydney and the Central Sydney Area Health Service. ASM cell characteristics were examined by immunofluorescence and light microscopy as previously described (25). All experiments were performed with cells between passages 4 and 8.
Real-Time Polymerase Chain Reaction
ASM cells from three to six patients without asthma and three to six patients with asthma were seeded in six-well plates for 24 h in 5% fetal bovine serum (FBS) at a density of 1 x 104 cells per cm2. Medium was then changed to 0.1% insulin, transferrin, and selenium (ITS) for 24 h before addition of TGF- (1 ng/ml), recombinant CTGF (250 ng/ml) (21), TGF- (1 ng/ml) and anti-CTGF antibody (0.5, 1, and 2 μg/ml) (rabbit polyclonal against mouse CTGF with cross reactivity to human CTGF), TGF- (1 ng/ml) and U0126 (5 μM) (26), LY294002 (3 μM) (27), SB203580 (10 μM), SB202474 (10 μM) (28), wortmannin (100 nM) (29–31), or dimethyl sulfoxide (0.001 or 0.006%). Total RNA was extracted from the cells at 8 or 24 h and real-time polymerase chain reaction performed for analysis of CTGF, fibronectin or collagen. Full details of the methods are provided in the online supplement.
ECM Protein Experiments
ASM cells from three to five patients without asthma and three to five patients with asthma were seeded in 96-well plates for 24 h in 5% FBS at a density of 1 x 104 cells/cm2. Medium was then changed to 0.1% ITS for 24 h before addition of TGF- (1 ng/ml) or CTGF (250 ng/ml) for 48 h. ECM free of cells was prepared by treatment with sterile hypotonic ammonium hydroxide. ECM proteins were measured by ELISA as previously described (32) using antibodies to fibronectin and collagen type I (33).
Immunohistochemistry
Human lung tissue was obtained from lung specimens resected for carcinoma or transplantation. Bronchial rings (2–5 mm in diameter and 3 mm in length) were dissected free from surrounding parenchymal tissue. The bronchial rings were incubated for 24 h in a 5% CO2 incubator at 37°C in 0.1% ITS in the presence and absence of TGF- (1 ng/ml) or CTGF (250 ng/ml). After 48 h, tissues were frozen in OTC embedding medium (Fronine Laboratory Supplies, Riverstone, Australia), sectioned on a cryostat, and immunohistochemistry performed using rabbit anti-mouse CTGF, mouse anti-human fibronectin, or mouse anti-human collagen I coupled with donkey anti-rabbit TRITC or horse anti-mouse Texas Red to detect the presence of CTGF, fibronectin, and collagen, respectively. To help identify the morphology of the tissue, hematoxylin and eosin staining was performed on serial sections.
Western Blotting
ASM cells from three patients without asthma and three patients with asthma were seeded in six-well plates for 24 h in 5% FBS at a density of 1 x 104 cells/cm2. Medium was then changed to 0.1% ITS for 24 h before addition of TGF- (1 ng/ml). Total protein was collected at time 0, and, after 10 and 30 min, Western blots were performed as described previously (34) using antibodies to total Smad-2 ([sc-6200; Santa Cruz Biotechnology, Inc., Santa Cruz, CA] diluted 1:500 in 2.5% skim milk Tween-phosphate buffered saline [T-PBS]) and phosphorylated Smad-2 (rabbit anti-phospho-smad2 [SER465/467] polyclonal antibody [Chemicon, Boronia, Victoria, Australia] diluted 1:1000 in 5% bovine serum albumin, Tween-tris buffered saline [BSA T-TBS]).
Total and Phosphorylated Akt ELISAs
ASM cells from one patient without asthma and one patient with asthma were seeded in six-well plates for 24 h in 5% FBS at a density of 1 x 104 cells/cm2. Medium was then changed to 0.1% ITS for 24 h before addition of TGF- (1 ng/ml). Total protein was collected at time 0 and after 10 min, 30 min, and 1 h in 100 μl Cell Extraction Buffer (10 mM Tris, pH 7.4, 100 mM NaCl, 1 mM ethylenediaminetetraacetic acid, 1 mM ethyleneglycol-bis-(-aminoethyl ether)-N,N'-tetraacetic acid, 1 mM sodium fluoride, 20 mM sodium pyrophosphate, 2 mM sodium orthovanadate, 1% Triton X-100, 10% glycerol, 0.1% sodium dodecyl sulfate, and 0.5% deoxycholate with Protease Inhibitor Cocktail Set III (Calbiochem, San Diego, CA) added at a concentration of 100 μl/ml. Samples were stored at –20°C until analysis. Total protein was determined using a bicinchonic acid protein assay kit (Sigma, St Louis, MO) as per the manufacturer's instructions. Total and phosphorylated Akt levels were measured by ELISA according to the manufacturer's instructions (Akt ELISA kit and Akt [pS473] ELISA kit; BioSource, Camarillo, CA). One microgram of total protein was loaded per well of the ELISA assay, and both standards and samples were assayed in duplicate. Standard curves were generated, and the duplicates averaged. The samples assayed all gave results within the linear part of the respective standard curve. Data generated for phosphorylated Akt were normalized against total Akt (ng/ml) using Microsoft Excel (Redmond, WA) software.
Statistical Analysis
Results from triplicate wells from each individual patient for cycle number for real-time reverse transcription polymerase chain reaction were meaned and an overall mean and SE were calculated for cycle number. Mean percentage change from 0.1% ITS values for ECM ELISA were obtained from patients with and without asthma. Analysis of variance using repeated measures and the Fisher protected least significant difference (PLSD) post test was performed on the results for real-time reverse transcription–polymerase chain reaction and ECM ELISAs. In all cases, a p value of 0.05 or less was considered significant.
RESULTS
Induction of Fibronectin and Collagen I by TGF-
TGF- (1 ng/ml) induced a significant fold increase in mRNA for fibronectin and collagen I in both the asthmatic and nonasthmatic ASM cells (Figures 1A and 1B). When results were expressed as a fold increase compared with 0.1% ITS, values for fibronectin mRNA were 3.6 ± 0.5 and 3.1 ± 0.4, and for collagen I were 3.1 ± 0.4 and 3.6 ± 0.5 (nonasthmatic [n = 16] and asthmatic [n = 10], respectively). TGF- (1 ng/ml) also induced a significant increase in deposited fibronectin and collagen I protein by both the asthmatic and nonasthmatic ASM cells (Figures 1C and 1D). Values for the protein production in response to TGF- (1 ng/ml) were 172.6 ± 24.9% (n = 5) and 175.9 ± 13.2% (n = 5) for fibronectin and 246.2 ± 37.0% (n = 3) and 245.0 ± 66.5% (n = 3) for collagen I (nonasthmatic and asthmatic, respectively; results expressed as a percentage of 0.1% ITS).
Incubation of bronchial tissues resected from a patient without asthma in TGF- (1 ng/ml) for 48 h resulted in the up-regulation of immunohistochemical staining for collagen I and fibronectin protein in the region of the airway smooth muscle, basement membrane, and generally in the interstitium (Figure 2).
Induction of Fibronectin and Collagen I by CTGF
Fibronectin and collagen I mRNA were significantly up-regulated by the addition of full-length recombinant CTGF protein in both the asthmatic and nonasthmatic ASM cells (Figures 3A and 3B). When results were expressed as a fold increase compared with 0.1% ITS, values for fibronectin mRNA were 2.1 ± 0.4 and 1.7 ± 0.3 and for collagen I 1.9 ± 0.6 and 1.5 ± 0.2 (nonasthmatic [n = 5] and asthmatic [n = 5], respectively).
In the presence of an anti-CTGF antibody (0.5–2 μg/ml), both fibronectin and collagen I mRNA induction by TGF- were significantly inhibited in both the nonasthmatic and asthmatic ASM cells (Figures 4A and 4B).
Incubation of bronchial tissues resected from a patient without asthma in CTGF (250 ng/ml) for 48 h resulted in the up-regulation of immunohistochemical staining for fibronectin and collagen I protein in the region of the airway smooth muscle, basement membrane, and generally in the interstitium (Figure 5).
TGF- Induction of Phosphorylated Smad-2
Total Smad-2 levels were not different at baseline between asthmatic (n = 3) and nonasthmatic (n = 3) ASM cells and were unaffected by treatment with TGF- for 10 or 30 min (data not shown). Phosphorylated Smad-2 levels were not different at baseline between asthmatic and nonasthmatic ASM cells; however, there was significant elevation of the levels above baseline after exposure to TGF- for 30 min. Furthermore, levels of phosphorylated Smad-2 were significantly higher in the asthmatic ASM cells in comparison to the nonasthmatic cells when exposed to TGF- for 10 and 30 min (Figure 6).
TGF-–mediated Signal Transduction
TGF- induction of fibronectin mRNA was significantly inhibited in both the nonasthmatic and asthmatic ASM cells by both the specific extracellular regulated kinase (ERK) inhibitor U0126 (60.0 ± 6.4% [n = 5] and 64.1 ± 10.3% [n = 4], respectively) and the p38 mitogen activated kinase (p38) inhibitor SB203580 (68.2 ± 6.9% [n = 6] and 56.1 ± 3.4% [n = 4], respectively, expressed as percentage of TGF- control; SB203580 inhibition compared with the effects of the inactive analog SB202474). When the involvement of PI3K in the TGF- induction of fibronectin mRNA was examined using the specific inhibitors LY294002 and wortmannin, only the asthmatic cells showed a significant reduction to 68.2 ± 6.0% with LY294002 (n = 4) and 61.6 ± 15.8 with wortmannin (n = 5; expressed as percentage of TGF- control; Figure 7A).
In contrast, the TGF- induction of collagen I mRNA was significantly inhibited in both the nonasthmatic and asthmatic ASM cells only by the specific ERK inhibitor U0126. Values for the nonasthmatic and asthmatic cells were 66.8 ± 6.9% (n = 6) and 64.0 ± 8.3% (n = 4), respectively (results expressed as percentage of TGF- control). As with the results with fibronectin mRNA, LY294002 induced a significant reduction in TGF- induction of collagen I mRNA (79.6 ± 7.4% [n = 6] expressed as percentage of TGF- control) only in the asthmatic ASM cells. Wortmannin inhibited TGF-–induced collagen I in four of the five patients with asthma analyzed, but only inhibited two of the four patients without asthma (Figure 7B).
When the signal transduction pathways involved in the TGF-–mediated induction of CTGF mRNA were examined, LY294002 was the only inhibitor to significantly inhibit CTGF mRNA in both nonasthmatic and asthmatic cells (62.7 ± 7.5% [n = 10] and 79.1 ± 8.4% [n = 12], respectively, expressed as percentage of TGF- control). Inhibition by wortmannin of responses to TGF-–mediated CTGF in patients with asthma fell into two distinct groups in that in half the patients, there was inhibition and in the other there was no inhibition (n = 4). Wortmannin caused a significant inhibition of the TGF-–induced CTGF in the nonasthmatic cells with values reduced to 67.5 ± 5.0% of control (n = 3). The ERK-specific inhibitor U0126 significantly inhibited TGF-–induced CTGF in nonasthmatic cells (80.9 ± 14.0% [n = 5], expressed as percentage of TGF- control), but not in asthmatic cells (n = 4; Figure 7C).
TGF- Induction of Phosphorylation of Akt
The ratio of phosphorylated Akt to total Akt was not different between asthmatic and nonasthmatic ASM cells at baseline (Figure 8). After 10 min stimulation with TGF- (1 ng/ml), there was greater induction of phosphorylated Akt in the asthmatic cells compared with the nonasthmatic cells (see Figure 8). This increased phosphorylation had returned to baseline by 30 min.
DISCUSSION
In this study, we have shown that TGF- induces the production of both fibronectin and collagen I in nonasthmatic and asthmatic ASM cells in culture and in sections of nonasthmatic bronchus. CTGF recombinant protein induced both fibronectin and collagen I mRNA in both nonasthmatic and asthmatic cells, an effect that was not different between the two cell types. The inhibition of fibronectin and collagen I mRNA induction by TGF- in the presence of an antibody to CTGF indicates that the TGF-–mediated induction of these two ECM proteins occurs in part by the autocrine effects of CTGF released from the cells. Differences between nonasthmatic and asthmatic ASM cells were observed in the signal transduction pathways involved in the induction of fibronectin and collagen by TGF-. The ERK and PI3K pathways were involved in collagen I induction in the asthmatic cells, whereas only ERK was involved in the nonasthmatic cells. In contrast ERK, PI3K, and p38 pathways were involved in fibronectin induction by TGF- in the asthmatic cells, whereas only ERK and p38 were involved in the nonasthmatic cells. This may suggest that the PI3K pathway is differentially up-regulated in the asthmatic cells. The role of PI3K in human ASM proliferation and migration has previously been reported, but this is the first time it has been implicated in ECM deposition. This is not the only evidence for differential activation/up-regulation of a signal transduction pathway in asthmatic ASM cells. We have previously reported that the ERK pathway may be differentially up-regulated in asthmatic ASM cells, possibly accounting for the increased rate of proliferation we have observed in this cell (35). Furthermore, the PI3K pathway was the only pathway of those studied involved in the TGF-–mediated induction of CTGF in the asthmatic cells, with both PI3K and ERK being involved in the nonasthmatic cells.
Fibronectin and collagen I are both ECM proteins that have been shown to be increased in the airways of patients with asthma. Collagen I is specifically increased in the region of the ASM. The present study suggests that TGF- may play a role in the production of these ECM proteins by the ASM. In the airways of patients with asthma, increases in the amount of TGF- have been observed (13). Although we did not observe differences between asthmatic and nonasthmatic ASM cells in fibronectin or collagen I mRNA or protein production, after stimulation with 1 ng/ml TGF-, the fact that there is more TGF- in the airways of patients with asthma would lead to greater production of the ECM proteins in vivo. Our results are consistent with the observation that TGF- increased fibronectin and procollagen I mRNA in human ASM cells in culture (11). Our study extends those findings to examine the effect of TGF- on fibronectin and collagen I production in asthmatic ASM cells and to characterize some of the signal transduction pathways involved.
Our observation that TGF- and CTGF lead to production of fibronectin and collagen in human ASM cells may have been a culture artifact; however, when fresh bronchial rings obtained from lung resections were incubated in TGF- or CTGF, increased protein for both fibronectin and collagen I in the region of the ASM was observed. This finding indicates that our observation in isolated ASM cells is unlikely to reflect that we studied a culture system.
The results of the present study suggest that CTGF is a downstream effector of TGF- in human nonasthmatic and asthmatic ASM cells. Recombinant CTGF alone induced both fibronectin and collagen I, a finding similar to that observed in a number of cell types, including normal rat kidney (NRK) fibroblasts (18), mesangial cells (36), and gingival fibroblasts (37). In our study, an antibody to CTGF inhibited the production of mRNA for both fibronectin and collagen I in response to TGF-. The degree of inhibition suggests that the TGF- induction of CTGF is only partially responsible for the TGF-–induced ECM protein production from the ASM cells. This finding is in agreement with studies by others in a variety of human and animal cells (18, 37–39); however, in contrast, TGF- induction of fibronectin, in rat renal fibroblasts, is totally CTGF-dependent (40).
Recent studies investigating the mechanism underlying TGF- signaling have indicated the importance of several pathways including the mitogen-activated kinases and PI3K (15, 16). In light of these observations, we were interested to determine if these pathways were also involved in TGF- signaling in ASM cells. The present study is the first to examine the signal transduction pathways involved in the TGF- induction of fibronectin and collagen in human ASM cells. The induction of fibronectin and collagen I share common pathways as well as using different pathways. Differences in signaling in the asthmatic and nonasthmatic cells were also observed. In the nonasthmatic cells, production of collagen I was ERK-dependent, whereas, in asthmatic cells, it was both ERK- and PI3K-dependent. The pathway involved in induction of collagen I expression appears to be cell type–specific (41–44). The activation of an alternative pathway in the asthmatic cells may represent an intrinsic difference between the asthmatic and nonasthmatic ASM cells. Further work is necessary to determine if the PI3K pathway is activated in asthmatic cells by stimuli other than TGF-. The induction of fibronectin by TGF- in the nonasthmatic cells was mediated via the ERK and the p38 pathways, whereas the asthmatic cells used both of these pathways and the PI3K pathway. Once again, the pathways that have previously been identified as being involved in TGF- modulation of fibronectin appear to be cell type–specific (45–47). In all of our inhibitor experiments, we were not able to completely inhibit the fibronectin or collagen I mRNA production with any one inhibitor, suggesting that the pathways we have identified are part of a complex process involved in the up-regulation of these genes. Other investigators have suggested that other signaling molecules including c-Jun N-terminal kinase (JNK) (48), phosphatidylcholine-specific phospholipase C, protein kinase C- (45), and the small guanosine triphosphate–binding protein Rac (41) are involved in the TGF-–mediated induction of collagen I or fibronectin. It is probable that these or other signaling molecules are working in concert with the signal transduction pathways we have identified in this study in the ASM cells.
In this study, we also examined the signal transduction pathways leading to the induction of CTGF after TGF- stimulation. Unlike the pathways leading to fibronectin and collagen, only PI3K was involved in the induction of CTGF in both the asthmatic and nonasthmatic ASM cells. In addition, we found that Akt phosphorylation (an event that occurs downstream of PI3K) occurred in both cell types; however, the ratio of phosphorylated to total Akt was greater in the asthmatic ASM cells. ERK was also involved in the induction of CTGF in nonasthmatic cells in agreement with the recent study by Xie and colleagues (31), although this study found that PI3K was not involved in the induction of CTGF in nonasthmatic ASM cells. It is possible that the different concentration of TGF- used in this study may explain these conflicting results. Our findings further emphasize that CTGF only partly mediates the TGF-–induced fibronectin and collagen I in human ASM cells. The involvement of PI3K in the induction of fibronectin and collagen (which may reflect the induction of CTGF before the induction of the ECM protein genes) in the asthmatic but not the nonasthmatic cells possibly indicates that CTGF is of more importance in the signaling events in the asthmatic ASM cells.
The mechanism by which TGF- induces CTGF is not clear at this stage, but may be cell type–specific, because conflicting results have been reported previously. In NIH 3T3 cells, protein kinase C and ERK were necessary for TGF-2 induction of CTGF (49, 50). Leask and colleagues also demonstrated that inhibition of JNK or mitogen-activated kinase p38 did not influence TGF-2–induced CTGF; however, overexpression of members of the JNK cascade resulted in suppression of the CTGF promoter (49). These authors suggest that a balance between ERK and JNK is necessary for TGF-–induced CTGF. In contrast, in the human lung fibroblast cell line, HFL-1, p38 and ERK were not involved in TGF-1–induced CTGF, but rather, as we found in our study, PI3K mediated this induction (51). This study showed that TGF- activated JNK-1 and JNK-2 and that the PI3K inhibitors were able to attenuate this activation demonstrating that JNK is downstream of PI3K. Whether this link exists in ASM cells is unclear; Xie and colleagues found JNK was involved in TGF-–mediated induction of CTGF, but PI3K was not (31). The reason for these conflicting reports is not clear, but may reflect the species difference in the source of the cells or the difference in the stimulus (i.e., TGF-2 vs. TGF-1). Because we were unable to inhibit the TGF-–mediated induction of CTGF by more than 40% with the inhibitors we studied, our results suggest that other pathways are also involved in the induction of CTGF.
We observed differential phosphorylation of SMAD-2 between the asthmatic and nonasthmatic ASM cells. Runyan and colleagues (52) found that cross talk between the SMAD and PI3K pathways enhanced TGF-–induced collagen I expression in human mesangial cells. In addition, Yi and colleagues (53) recently reported that TGF- activation of PI3K was regulated by SMADs. Similarly, Xie and colleagues reported that inhibition of ERK and JNK inhibited TGF-–induced SMAD2/3 phosphorylation in nonasthmatic ASM cells (31). The increased phosphorylation of SMAD-2 in the asthmatic cells may be linked to the involvement of PI3K signaling in these cells, but not in the nonasthmatic cells. Further investigations are necessary to determine the interdependence of the SMAD (for which there are no commercial inhibitors available) and mitogen-activated kinases/PI3K pathways in TGF-–induced CTGF and ECM genes in asthmatic ASM cells. Kucich and colleagues reported that phosphatidylcholine-specific phospholipase C, protein kinase C, and one or more tyrosine kinases were involved in TGF-–mediated induction of CTGF; it is also possible that one or more of these molecules is working in concert with PI3K in the ASM cells. Further work is needed to understand the complex interplay of these multiple pathways in the induction of CTGF and the role this plays in the regulation of ECM protein genes in the airways.
Our studies have identified several pathways that are involved in the TGF-–mediated induction of the ECM proteins fibronectin and collagen. Of major importance is the potential difference we have noted in the signaling pathways between the asthmatic and nonasthmatic cells. It may be possible to selectively target TGF-–induced PI3K signaling in asthmatic ASM cells without disrupting the other signaling pathways that lead to other important functions of TGF-.
Acknowledgments
The authors acknowledge the collaborative effort of the cardiopulmonary transplant team and the pathologists at St. Vincent's Hospital, Sydney, Australia. They thank Dr. Greg King from the Woolcock Institute of Medical Research, Royal Prince Alfred Hospital, Sydney, Australia, and Dr. Melissa Baraket from the Department of Pharmacology, The University of Sydney, Sydney, Australia, for the supply of lung biopsies.
FOOTNOTES
Supported by funds from the National Health and Medical Research Council (NH&MRC) of Australia, and by an NH&MRC Peter Doherty Fellowship 165722 (J.K.B.).
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
Originally Published in Press as DOI: 10.1164/rccm.200406-703OC on September 22, 2005
Conflict of Interest Statement: None of the authors have a financial relationship with a commercial entity that has an interest in the subject of the manuscript.
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