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American Journal of Respiratory and Critical Care Medicine Vol 167. pp. 71-77, (2003)
© 2003 American Thoracic Society

Original Article

Expression of Connective Tissue Growth Factor in Asthmatic Airway Smooth Muscle Cells

Janette K. Burgess*, Peter R. A. Johnson*, Qi Ge, Wendy W. Au, Maree H. Poniris, Brent E. McParland, Greg King, Michael Roth and Judith L. Black

Department of Pharmacology, University of Sydney; and Woolcock Institute of Medical Research, Royal Prince Alfred Hospital, Sydney, Australia

Correspondence and requests for reprints should be addressed to Dr. Janette Burgess, Ph.D., Respiratory Research Group, Department of Pharmacology, Bosch Building, D05 University of Sydney, Sydney NSW, Australia, 2006. E-mail: janette{at}pharmacol.usyd.edu.au


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
There is strong evidence to implicate transforming growth factor-ß in the remodeling that occurs in asthma, as levels are increased in bronchial lavage fluid and gene expression is increased in bronchial tissue. Transforming growth factor-ß is also known to increase the release of collagen from airway smooth muscle. Here we identify for the first time a possible mechanism for the effects of transforming growth factor-ß. Transforming growth factor-ß specifically induces mRNA and protein for connective tissue growth factor in airway smooth muscle, and moreover, we report that the connective tissue growth factor response is greater in airway smooth muscle cultured from patients with asthma compared with patients without asthma. This occurs at both the level of mRNA (37.53 ± 11.62- and 13.59 ± 3.12-fold increase at 24 hours compared with time 0, respectively, p < 0.02) and protein production (67.57 ± 27.80- and 3.58 ± 0.6-fold increase at 24 hours compared with time 0, respectively, p < 0.03). The differential connective tissue growth factor response to transforming growth factor-ß in asthmatic airway smooth muscle identifies a potential role for connective tissue growth factor in the remodeling that is characteristic of severe persistent asthma.

Key Words: asthma • extracellular matrix • transforming growth factor-ß


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Airway wall remodeling is one of the key features of asthma. The extracellular matrix provides the mechanical support required for airway structure and function but also has the potential to influence a variety of functions of the embedded cells, including migration, proliferation, and differentiation (1, 2). Part of the remodeling process involves the alteration of the local composition of the extracellular matrix within the airways. In asthmatic airways, the deposition of collagen I, III, and V, fibronectin, tenascin, hyaluronan, versican, and laminin {alpha}2 and ß2 are reported to be increased, whereas collagen IV and elastin are decreased (3–5). The underlying mechanisms resulting in the altered extracellular matrix deposition are currently not well understood.

One protein that has been suggested to play a role in the extracellular matrix remodeling that occurs during fibrosis and scarring is connective tissue growth factor (CTGF). CTGF is a cysteine-rich 38-kD protein that was originally identified as a growth factor secreted by human umbilical vein endothelial cells (6). CTGF upregulates gene expression of collagen I and fibronectin, which leads to increased protein production in human lung fibroblasts (7) and vascular smooth muscle cells (8).

Transforming growth factor-ß (TGF-ß) selectively upregulates CTGF mRNA and protein synthesis in fibroblasts, whereas other growth factors such as platelet-derived growth factor (PDGF) have no effect (9). TGF-ß has been suggested to play a role in airway remodeling in asthma (10–12) and is produced by a range of cell types, including platelets, macrophages, epithelial cells, and airway smooth muscle cells, as an inactive form that can be targeted to the extracellular matrix (13). Plasmin is able to regulate the conversion and the release of the biologically active TGF-ß from the extracellular matrix, which is subsequently able to induce airway smooth muscle to synthesize procollagen I in an autocrine manner (11). TGF-ß has also been observed to induce proliferation of airway smooth muscle (14) and to enhance the production of extracellular matrix proteins (11, 15) by airway smooth muscle.

In asthma, it has been reported that the concentration of TGF-ß in bronchial lavage fluid is higher than in normal control subjects (12), and TGF-ß gene expression has been observed to be increased in bronchial tissue from subjects with asthma (16–18). Increased immunoreactivity for TGF-ß has been demonstrated in bronchial biopsies and submucosal eosinophils from subjects with asthma (16, 18). Many of the observed effects of TGF-ß in injury and fibrosis, such as cell proliferation, migration to site of injury, and extracellular matrix synthesis, have been shown to be mediated via CTGF (9, 19, 20). The possibility that the TGF-ß–mediated airway remodeling effects may be occurring via CTGF has not been examined previously. In this study, we tested the hypothesis that TGF-ß might induce CTGF expression and release in human airway smooth muscle cells and compared the effects of TGF-ß in asthmatic and nonasthmatic airway smooth muscle cells.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals
Dulbecco's modified Eagle's medium (DMEM), insulin transferrin selenium (ITS), Dulbecco's phosphate-buffered saline, penicillin, streptomycin, amphotericin B, trypan blue, (Life Technologies, Heidelberg, Australia), and fetal bovine serum (FBS) (Commonwealth Serum Laboratories, Melbourne, Australia) were obtained from the sources given in parentheses.

Antibodies and Recombinant Proteins
Tetramethylrhodamine isothiocyanate-conjugated goat anti-rabbit immunoglobulin G (Sigma, St. Louis, MO), rabbit polyclonal against mouse CTGF (CTGF-ab6992) with cross-reactivity to human CTGF (Abcam Ltd., Cambridge, UK), and anti–TGF-ß antibody (R&D Systems, Minneapolis, MN) were purchased as indicated. The goat anti-rabbit horseradish peroxidase–conjugated secondary antibody, Hoechst nuclear stain #33,258 and recombinant human TGF-ß and PDGF were from Sigma.

Cell Culture
Nonasthmatic airway smooth muscle was obtained from bronchial airways of three patients undergoing resection for lung transplantation and two deep endobronchial biopsies obtained by flexible bronchoscopy, and asthmatic airway smooth muscle was obtained from one lung donation, one patient dying in status asthmaticus, and four deep endobronchial biopsies by methods previously described (21–24). The details about the patients are shown in Table 1 . Approval for all experiments with human lung was provided by the Human Ethics Committee of the University of Sydney and the Central Sydney Area Health Service, and all patients provided written informed consent. Airway smooth muscle cell characteristics were determined by immunofluorescence and light microscopy (25).


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  		TABLE 1. Patient characteristics

 
Stimulation with TGF-ß
Airway smooth muscle cells were seeded in six-well plates at 1 x 104 cells/cm2 in 5% FBS DMEM for 24 hours before being synchronized for 24 hours in 0.1% ITS in DMEM. For stimulation, TGF-ß (1 ng/ml) or PDGF (10 ng/ml) was added to the cells for 15 minutes to 24 hours. To confirm the role of TGF-ß, we performed experiments in the presence of a neutralizing anti–TGF-ß antibody (1.2 ng/ml). Control cells were maintained in 0.1% ITS in DMEM. At the completion of each incubation, cells were washed in ice-cold phosphate-buffered saline and kept on ice until lysis in protein extraction buffer (20 mM Tris, pH 7.4, 1 mM ethylenediaminetetraacetic acid, 1 mM benzamidine, 1 mM PMSF, 1 mM Aprotinin, and 10% Triton X-100). Cells were scraped from the culture wells with multiple pipetting and then stored at -20°C until analysis. Cells for RNA analysis were lyzed and stored at -20°C until extraction. Total RNA was extracted from airway smooth muscle cells using the RNeasy Mini Kit (Qiagen, Clifton Hill, Australia) according to the manufacturer's instruction. After extraction, samples were eluted in 30-µL RNase-free water and stored at -20°C until use.

Laser Capture Microdissection
Nonasthmatic lung tissue containing bronchial segments was mounted in optimum cutting temperature compound (OCT) (Miles, Elkhart, IN) and snap frozen in liquid nitrogen–cooled isopentane before storage at -80°C. The tissues were brought to -20°C and 5- to 6-µm sections cut before being placed on plain glass slides at room temperature and immediately fixed. Sections were prepared for laser capture microdissection. Additional detail on the preparation of the sections is provided in the online supplement. Airway smooth muscle bundles were captured onto the CapSure High Sensitivity LCM caps and stored at -70°C in the presence of 10 to 20 µL lysis buffer until RNA was isolated using the Total RNA Microprep kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions.

Real-Time Reverse Transcription-Polymerase Chain Reaction
Real-time reverse transcription-polymerase chain reaction (RT-PCR) was performed for the detection of CTGF mRNA (CTGF forward primer [5'-TGTGTGACGAGCCCAAGGA-3'], reverse primer [5'-TCTGGGCCAAACGTGTCTTC-3'], and internal probe [FAM-5'-TGGTTGGGCCTGCCCTCGC-3'-TAMRA]) using the TaqMan One-Step RT-PCR Master Mix Reagents Kit (PE Applied Biosystems, Foster City, CA). For precise quantitative analysis of gene expression, the predeveloped TaqMan Assay Reagents (Endogenous Control Ribosomal RNA Control [18S rRNA]) (PE Applied Biosystems) was included in the RT-PCR reactions. Additional detail about the RT-PCR is provided in the online supplement. Data from the reaction were collected and analyzed by the complementary computer software (26).

Immunohistochemistry
We performed immunohistochemistry to detect the presence of CTGF on human airway smooth muscle cells grown on glass coverslips for 5 days in 10% FBS DMEM followed by 24 hours with or without TGF-ß (1 ng/ml) as described previously (27). CTGF was detected using the rabbit polyclonal antibody raised against mouse CTGF (CTGF-ab6992) with cross-reactivity to human CTGF followed by the goat anti-rabbit horseradish peroxidase–conjugated secondary antibody. Hoechst was used to stain the nuclei of all cells present on the coverslip.

Western Blot
Cellular proteins were size fractionated on 10% polyacrylamide gels, transferred to polyvinylidene difluoride membranes, and blocked overnight in 5% (wt/vol) skim milk solution as described previously (23). The membranes were incubated with a rabbit polyclonal anti-CTGF antibody (1 in 5,000 in skim milk solution) for 1 hour before washing and incubation with secondary antibody (1 in 40,000 in skim milk solution) for 1 hour. Immunoblot detection was performed using a supersignal west dura extended duration substrate kit (Pierce, Rockford, IL), and bands were analyzed using a 440F Kodak imaging system and software.

Statistical Analysis
In real-time PCR experiments, the number of cycles needed to attain a threshold fluorescence set to lie on the exponential part of the amplification plot was determined. Results for CTGF were normalized against those obtained for 18S and were expressed as a cycle difference (basal levels) or a fold increase (TGF-ß stimulated). Results from triplicate wells from each individual patient were meaned, and an overall mean and SEM were calculated for cycle number. Analysis of variance using repeated measures and the Fisher protected least square differences (PLSD) post-test was performed on the results for real-time RT-PCR and mean densitometric values for Western blots. Factorial analysis of variance was used to compare CTGF mRNA and protein production curves generated from asthmatic and nonasthmatic cells. In all cases, a p value of less than 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Detection of CTGF mRNA in Airway Smooth Muscle
The amplification of CTGF mRNA in RNA isolated from asthmatic and nonasthmatic airway smooth muscle in culture using real-time RT-PCR identified for the first time the presence of mRNA encoding CTGF in both cells types. In a separate series of experiments to control for culture artifact, we used laser capture microdissection to isolate airway smooth muscle cells from lung sections obtained from three individuals, and subsequent amplification of the CTGF mRNA by real-time RT-PCR demonstrated that the CTGF mRNA was present in airway smooth muscle in the lung. A representative graph of RNA from both captured and cultured nonasthmatic airway smooth muscle cells is shown in Figure 1 .



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  		Figure 1. Real-time RT-PCR detection of CTGF gene expression in nonasthmatic airway smooth muscle cells. CTGF-specific Taqman primers and probe were used with 100-ng total RNA isolated from nonasthmatic airway smooth muscle cells in culture grown in 5% FBS in DMEM for 7 days (squares) or total RNA isolated from 1,000 airway smooth muscle cells obtained from a nonasthmatic lung section using laser capture microdissection (Xs). {Delta}Rn represents the changes in fluorescence. The graph is representative of the results seen in three separate experiments.

 
Detection of CTGF Protein in Airway Smooth Muscle
To determine whether CTGF protein was detectable, airway smooth muscle cells were grown on glass coverslips for immunohistochemistry. Cells were stained with the secondary antibody alone as the fluorescence control (Figure 2A) . The level of fluorescence in unstimulated airway smooth muscle cells was above the background level of fluorescence, indicating the presence of CTGF in these cells (Figure 2B). As TGF-ß has been reported to induce upregulation of CTGF in other cell types, we investigated the effect of this growth factor on CTGF in airway smooth muscle cells. After the airway smooth muscle cells had been stimulated with TGF-ß for 24 hours, there was a significant increase in the level of fluorescence detected (Figure 2C). The staining was not located in the nuclear compartments of the cells but rather was in the cytoplasmic regions.



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  		Figure 2. Detection of CTGF protein in airway smooth muscle cells in culture. Airway smooth muscle cells were allowed to adhere to glass coverslips for 5 days in 10% FBS in DMEM before being stimulated in 10% FBS DMEM with or without TGF-ß (1 ng/ml) for a further 24 hours. Omission of the primary antibody was used as a control (A). Cells incubated in the absence (B) or presence (C) of TGF-ß were stained with rabbit anti-CTGF primary and secondary antibody conjugated with tetramethylrhodamine isthiocyanate (red staining). Hoechst nuclear stain was used to identify the location of the cells in the field of view.

 
Stimulation of CTGF mRNA with TGF-ß
TGF-ß has been reported to induce upregulation of CTGF mRNA in fibroblasts and vascular smooth muscle cells, and thus, we determined the effect of this growth factor on CTGF mRNA in airway smooth muscle cells. To avoid the influence of serum induction of CTGF expression, the experiments were performed in DMEM supplemented with 0.1% ITS over a 24-hour time course, and the induction of CTGF gene expression by 0.1% ITS was measured at each of the time points to ensure that it was not contributing significantly to the expression observed in the presence of other stimuli (Figure 3A) . Expression of CTGF in the absence of TGF-ß (time 0) was not significantly different in the asthmatic and the nonasthmatic cells (6.4 ± 0.14 and 6.6 ± 0.3 cycles difference between CTGF and 18S rRNA, respectively). The expression of CTGF mRNA was upregulated by TGF-ß in both the asthmatic and the nonasthmatic cells over the 24-hour time course. Significantly enhanced expression, compared with expression at time 0, was observed after 8 and 24 hours in both the asthmatic (40.32 ± 8.88-fold increase, p < 0.01, and 37.53 ± 11.62-fold increase, p < 0.01) and the nonasthmatic cells (11.89 ± 5.58-fold increase, p < 0.05, and 13.59 ± 3.12-fold increase, p < 0.01), respectively (Figure 3B). The overall increase in CTGF mRNA expression in response to TGF-ß was significantly greater in the asthmatic cells compared with the nonasthmatic cells (p < 0.02) (Figure 3B). Preincubation of the TGF-ß with an anti–TGF-ß antibody significantly reduced the TGF-ß–mediated CTGF mRNA induction observed at 24 hours in the asthmatic cells (37.5 ± 17.19% TGF-ß response, p < 0.05) and at 0.5, 2, and 24 hours in the nonasthmatic cells (38.82 ± 8.1% TGF-ß response, p < 0.05, 29.67 ± 7.06% TGF-ß response, p < 0.01, and 33.0 ± 12.66% TGF-ß response, p < 0.01, respectively) (Figure 3C).



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  		Figure 3. Stimulation of CTGF mRNA with TGF-ß. Airway smooth muscle cells were allowed to adhere for 24 hours in 5% FBS in DMEM before being synchronized for 24 hours in 0.1% ITS in DMEM. Control cells were maintained in 0.1% ITS in DMEM throughout the time course (A). Cells were stimulated with TGF-ß (1 ng/ml) (B) for durations from 15 minutes to 24 hours and total RNA collected. Real-time RT-PCR was performed, and cycle thresholds were normalized to the expression of the control (18S rRNA) gene in each sample. The results are expressed as the fold change in CTGF mRNA expression relative to the expression at time 0 hour in asthmatic (solid bars) (n = 5) and nonasthmatic (open bars) (n = 5) cells. The TGF-ß was preincubated with an anti–TGF-ß antibody for 1 hour before addition to the cells and resultant CTGF gene expression expressed as a percentage of the expression seen in the presence of TGF-ß alone at each time point (C). Expression was significantly different from time 0 *p < 0.05, **p < 0.01; asthmatic curve was significantly different from the nonasthmatic curve #p < 0.02.

 
Stimulating the airway smooth muscle cells with PDGF resulted in minimal increase of CTGF message compared with the levels seen in the presence of TGF-ß. A small significant response was observed at 8 hours in the nonasthmatic cells (3.18 ± 1.27-fold increase, p < 0.05), and the small fold change observed at 2 hours was significant in the asthmatic cells (4.94 ± 0.98-fold increase, p < 0.01). The difference in the response between the asthmatic and the nonasthmatic cells after treatment with PDGF was found to be significant (p < 0.02) (data not shown).

Detection of CTGF Protein Induction by TGF-ß in Airway Smooth Muscle Cells
To determine whether the differences observed at the message level were translated into differences at the protein level, we measured the level of CTGF protein in the asthmatic and nonasthmatic airway smooth muscle cells by Western blot (Figure 4) . The amount of protein present in each sample was determined as the densitometric density and was expressed as a fold increase in the densitometric density of the protein compared with the densitometric density detected at time 0 hour. Similar to the mRNA results, the amount of protein stimulated by the 0.1% ITS alone was measured as the control, and no significant increase in the amount of CTGF protein was stimulated by ITS in either the asthmatic or the nonasthmatic cells (Figure 5A) .



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  		Figure 4. Western blot for CTGF after TGF-ß stimulation. Airway smooth muscle cells were allowed to adhere for 24 hours in 5% FBS in DMEM before being synchronized for 24 hours in 0.1% ITS in DMEM. Cells were stimulated with TGF-ß (1 ng/ml) for durations from 30 minutes to 24 hours and total protein collected. Western blot analysis was performed using chemiluminescent detection. The higher molecular weight band seen at time 0 was nonspecific. The blots are representative of the results seen for n = 5 nonasthmatics (A) and n = 6 asthmatics (B).

 


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  		Figure 5. Level of CTGF protein after stimulation of airway smooth muscle with TGF-ß. Airway smooth muscle cells were allowed to adhere for 24 hours in 5% FBS in DMEM before being synchronized for 24 hours in 0.1% ITS in DMEM. Control cells were maintained in 0.1% ITS in DMEM throughout the time course (A). Cells were stimulated with TGF-ß (1 ng/ml) (B) for durations from 15 minutes to 24 hours and total protein collected. Western blot analysis was performed and the results are expressed as the fold expression relative to the expression at time 0 hour in asthmatic (solid bars) (n = 6) and nonasthmatic (open bars) (n = 5) cells. The TGF-ß was preincubated with an anti–TGF-ß antibody for 1 hour before addition to the cells and resultant CTGF protein detected expressed as a percentage of the protein seen in the presence of TGF-ß alone at each time point (C). Expression was significantly different from time 0 *p < 0.05, **p < 0.01; the asthmatic curve was significantly different from nonasthmatic curve #p < 0.02.

 
In the presence of TGF-ß, a significant induction of CTGF protein expression was observed in both the asthmatic and the nonasthmatic cells over the 24-hour time course. The asthmatic cells showed significantly greater expression at 24 hours (67.57 ± 27.8-fold increase, p < 0.01) and the nonasthmatic cells at 2, 8, and 24 hours (2.65 ± 0.55-fold increase p < 0.02, 2.49 ± 0.35-fold increase, p < 0.05, and 3.58 ± 0.6-fold increase, p < 0.01, respectively) compared with the expression observed at time 0. Overall, the asthmatic cells showed a significantly greater response to TGF-ß than the nonasthmatic cells (p < 0.03) (Figure 5B). When the TGF-ß was preincubated with a neutralizing TGF-ß antibody, the TGF-ß–mediated induction of CTGF protein expression was markedly downregulated in both the asthmatic (28.76 ± 14.4% TGF-ß response, p < 0.05) and the nonasthmatic cells (25.05 ± 7.91% TGF-ß response, p < 0.01) at 24 hours (Figure 5C). PDGF did not stimulate an increase in the level of CTGF protein in either the asthmatic or the nonasthmatic cells, with the small response seen in the asthmatic cells not reaching significance (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This is the first study to identify human airway smooth muscle cells as a source for CTGF mRNA and protein. We have shown the CTGF protein in airway smooth muscle is specifically inducible by TGF-ß and is not induced by PDGF. In addition, we report for the first time an increase in CTGF mRNA and protein expression in asthmatic airway smooth muscle compared with nonasthmatic airway smooth muscle.

A significant induction of CTGF mRNA synthesis after TGF-ß stimulation of the airway smooth muscle cells was observed after 8 hours. The fold change in expression of CTGF message was significantly greater in the asthmatic cells than in the nonasthmatic cells. The maximal expression of mRNA occurred somewhere between 8 and 24 hours. The asthmatic cells showed greatest expression at 8 hours, but the nonasthmatic cells were still increasing in mRNA synthesis at 24 hours, indicating that asthmatic airway smooth muscle cells may have a shortened or more sensitive TGF-ß signaling cascade or may express higher numbers of TGF-ß receptors.

The level of protein expression detected was maximal in both cell types at 24 hours. It is possible that the protein levels may continue to increase beyond the 24-hour time point, which was the last time point in this study. There is a possibility that a gene can be activated during culture, and our finding of the presence of CTGF may have represented a culture artifact. To control for this, we captured airway smooth muscle cells from sections of nonasthmatic lung and indeed found that CTGF was present in isolated muscle bundles. This demonstrates that the CTGF gene is expressed in this specific cell type in the human lung, and this raises the possibility that it may contribute to the lung structure.

TGF-ß is a multifunctional protein that is one of the most potent regulators of inflammation and connective tissue synthesis (28). It has previously been demonstrated to induce fibroblasts, in culture, to proliferate, and to synthesize extracellular matrix proteins such as collagen {alpha}1, fibronectin, and proteoglycans (29, 30). TGF-ß also induces proliferation and collagen {alpha}1 release from airway smooth muscle cells in vitro (10, 11, 14, 15). Many of the mitogenic and extracellular matrix synthesis–promoting effects of TGF-ß on connective tissue cells are mimicked by CTGF (6, 31, 32), but the presence of this growth factor in airway smooth muscle cells has not been examined previously.

In the airway smooth muscle cells, the levels of CTGF, in response to TGF-ß stimulation, were markedly decreased when cells were preincubated with an anti–TGF-ß antibody. The small response seen in the presence of the antibody may be due to the fact that we did not achieve a stoichiometric balance between the antibody and the TGF-ß concentrations.

Only a small increase in CTGF mRNA synthesis in response to PDGF was observed in the airway smooth muscle cells, but this was not reflected at the protein level in the airway smooth muscle cells, which is consistent with findings in other cell types (9, 31), viz that the induction of CTGF is specific to TGF-ß.

As is true for fibroblasts, the TGF-ß–mediated effects in airway smooth muscle could be occurring via CTGF. Cohen and colleagues recently reported that insulin growth factor binding protein 3 (IGFBP3)–mediated TGF-ß–induced airway smooth muscle cell growth (14). They observed an increase in IGFBP3 mRNA 24 hours after stimulation with TGF-ß, and observed an enhanced release of IGFBP3 from the airway smooth muscle 72 hours after TGF-ß stimulation; however, they did not determine whether the increase in IGFBP3 mRNA was dependent on the de novo synthesis of mRNA, prolonged survival of mRNA, or enhanced translation. It is possible that CTGF is also contributing to this TGF-ß–mediated cell growth in airway smooth muscle. CTGF could be an upstream event involved in the signaling process leading to increased IGFBP3 mRNA and protein, as we observed the increases in CTGF much earlier (mRNA by 8 hours and the protein was significantly increased by 24 hours after TGF-ß stimulation) than the increases reported for IGFBP3. Alternatively, the CTGF-mediated effects could be acting independently of the IGFBP3 events.

TGF-ß induces an autocrine increase in the synthesis of collagen I in bovine airway smooth muscle cells (11). As CTGF has been reported to increase collagen {alpha}I mRNA and protein release from fibroblasts (7), the TGF-ß–mediated increase in collagen I in airway smooth muscle could also be mediated via CTGF. The upregulation of CTGF in airway smooth muscle cells may also be involved in controlling the expression of fibronectin (as seen in fibroblasts [7] and vascular smooth muscle cells [8]), and other extracellular matrix proteins that have yet to be examined. CTGF has recently been shown to increase the activity of matrix metalloproteinase 2 by altering the mRNA level of matrix metalloproteinase 2 in vascular smooth muscle cells (33). Under physiologic conditions, the expression of matrix metalloproteinases and their specific inhibitors, the tissue inhibitors of metalloproteinases, is highly coordinated at the level of gene expression (34). This balanced expression guarantees normal tissue structure and organ function and prevents both excessive extracellular matrix deposition and increased extracellular matrix degradation. In the lung, distortion of this balance is associated with various inflammatory lung diseases and lung cancer (35, 36).

We have previously shown that the profile of extracellular matrix proteins released from nonasthmatic cells can be modified by the allergic process (37). In addition, we have demonstrated that the asthmatic airway smooth muscle cells release a different profile of extracellular matrix proteins compared with nonasthmatic airway smooth muscle cells (38). The differential expression and the faster induction of CTGF observed in the asthmatic cells in this study could play a key role in the regulation, production, and degradation of the different extracellular matrix proteins released by asthmatic airway smooth muscle cells.

TGF-ß is increased in the bronchoalveolar lavage fluid of patients with asthma compared with normal control subjects, and the levels of TGF-ß were further increased in the patients with asthma after an allergen challenge (12). In addition, increased TGF-ß1 gene expression has been found in submucosal eosinophils from asthmatic airways (16, 17), and increased immunoreactivity was observed in biopsies from asthmatics and chronic bronchitic individuals compared with control individuals (18). Together with our results in the current study, it can be assumed that these increases in the level of TGF-ß present in the asthmatic airways would lead to further induction of CTGF expression. Whether this would lead to the presence of CTGF protein in human airways is not known.

Our study supports this hypothesis by demonstrating significantly increased production of CTGF mRNA and protein in asthmatic airway smooth muscle compared with nonasthmatic airway smooth muscle after exposure to TGF-ß. The reasons for this altered response are not known at this stage but could result from the asthmatic cells being more sensitive to the TGF-ß stimulus. This could be mediated through a greater number of TGF-ß receptors on the cell surface. Alternatively, one of the signaling pathways negatively controlling the expression of CTGF may be impaired in the asthmatic cells. One potential mechanism that may be disrupted in the asthmatic cells is the prostaglandin E2–mediated regulation of CTGF expression. Prostaglandin E2 can block the TGF-ß–mediated increase in CTGF mRNA and subsequent protein production in fibroblasts (39). TGF-ß is known to stimulate prostaglandin E2 release from human airway smooth muscle (40). We have recently shown that asthmatic airway smooth muscle cells release significantly less prostaglandin E2 than nonasthmatic airway smooth muscle cells (Linda Chambers, personal communication). This may indicate that a significant negative feedback mechanism for the control of CTGF expression is impaired in the asthmatic cells. Without the negative control by prostaglandin E2, the CTGF-mediated effects, such as cell growth and production of extracellular matrix proteins, could contribute significantly to the airway remodeling observed in asthma.

Until the recent study published by our group (24), it has not been possible to study asthmatic airway smooth muscle cells because of their lack of availability to determine which control mechanisms are altered in these cells. However, previous studies have identified differences in the adenylate cyclase system in asthmatic peripheral blood mononuclear cells compared with nonasthmatic peripheral blood mononuclear cells after allergen challenge, with consistent failure to elevate cAMP levels in response to specific stimuli (41).

Interestingly, elevation of cAMP levels within NRK fibroblasts inhibits TGF-ß–induced CTGF gene induction (42, 43) and subsequent collagen synthesis (19). Therefore, the altered regulation of the adenylate cyclase system identified in the asthmatic peripheral blood mononuclear cells could also be playing a role in CTGF regulation in airway smooth muscle contributing to the increased production of CTGF mRNA and protein in asthmatic airway smooth muscle compared with nonasthmatic airway smooth muscle.

In summary, this is the first study to identify CTGF mRNA and protein in airway smooth muscle cells in the human lung and most importantly to observe an enhanced responsiveness of CTGF gene expression in response to TGF-ß in asthmatic airway smooth muscle. The differential CTGF response identifies a potential role for CTGF in the remodeling, which is characteristic of persistent asthma. Further studies are necessary to understand the role of CTGF in the pathogenesis of asthma and therefore determine its importance as a potential future therapeutic target.


   Acknowledgments
 
The authors acknowledge the collaborative effort of the cardiopulmonary transplant team and the pathologists at St. Vincent's Hospital, Sydney, Australia.


   FOOTNOTES
 
Supported by the National Health and Medical Research Council, Australia, and a 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 online at www.atsjournals.org

* These authors made equal contributions to this manuscript. Back

Received in original form May 13, 2002; accepted in final form September 9, 2002


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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