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Acta Biochim Biophys
Sin 2006, 38: 53-57 |
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doi:10.1111/j.1745-7270.2006.00130.x |
Connective Tissue Growth Factor Expression in Human Bronchial Epithelial Cells
Amrita DOSANJH*
Department of Molecular
and Experimental Medicine, the Scripps Research Institute, La Jolla 92122, USA
Received: September
23, 2005
Accepted: November
3, 2005
*Corresponding
author: E-mail, [email protected]
Abstract������� Connective tissue growth factor (CTGF) is a cysteine-rich protein that promotes extracellular matrix deposition. CTGF is selectively induced by transforming growth factor b and des-Arg kallidin in lung fibroblasts and increases steady-state mRNA levels of a type I collagen, 5a-integrin and fibronectin in fibroblasts. Bronchial epithelial cells have been proposed to functionally interact with lung fibroblasts. We therefore investigated if bronchial epithelial cells are able to synthesize CTGF. Human bronchial epithelial cells were grown to subconfluence in standard growth media. Proliferating cells grown in small airway growth media were harvested following starvation for up to 24 h. Expression of CTGF transcripts was measured by PCR. Immunocytochemistry was also completed using a commercially available antibody. The cells expressed readily detectable CTGF transcripts. Starvation of these cells resulted in a quantitative decline of CTGF transcripts. Direct sequencing of the PCR product identified human CTGF. Immunocytochemistry confirmed intracellular CTGF in the cells and none in negative control cells. We conclude that bronchial epithelial cells could be a novel source of CTGF. Bronchial epithelial cell-derived CTGF could thus directly influence the deposition of collagen in certain fibrotic lung diseases.
Key words������� bronchial epithelial cell; connective tissue growth factor (CTGF); collagen deposition; lung remodeling
A wide variety of growth factors could be involved in the remodeling of airway tissue in diseases such as asthma. Collagen deposition and lung remodeling are now recognized features of a subset of asthmatic patients [1]. Connective tissue growth factor (CTGF) is an important regulator of collagen deposition that has not been extensively studied in the lung. CTGF was first isolated from human umbilical artery endothelial cells, and is a member of the CCN gene family, which contains insulin growth factor binding domains. CTGF is a cysteine-rich protein, with a molecular mass of approximately 38 kDa, that is transcriptionally regulated by transforming growth factor b (TGF-b) and other factors, such as des-Arg kallidin. CTGF promotes fibroblast proliferation, migration, adhesion and extracellular matrix (ECM) formation, and its overproduction might play a major role in pathways that lead to fibrosis [2,3].
In some asthmatics, the deposition of ECM and remodeling of the airways is observed, which leads to worsening clinical symptoms despite treatment. Epithelial activation and regulation of fibroblasts are proposed to be important in asthmatic remodeling. To better understand the process of remodeling in lung disease, it is important to identify the bronchial epithelial cell-derived factors that regulate the deposition of collagen and ECM in the lung. CTGF has been well studied in connective tissue and fibroblasts [4,5]. Although some insulin-like growth factors (IGF) have been identified in epithelial cells, the expression of CTGF, in particular, has not been described in bronchial epithelial tissue or cells.
We hypothesize that bronchial epithelial cells might produce CTGF, and thus can directly regulate the deposition of collagen and ECM by lung fibroblasts, which are in close proximity to bronchial epithelial cells.
Materials and Methods
Cell culture and RNA isolation
Human bronchial epithelial cells (HBEC) from two separate donors (Clonetics, San Diego, USA) were incubated and grown in small airway growth media (SAGM; Clonetics) in room air with 5% CO2 at 37 �C under sterile conditions. HBEC were grown to approximately 75% subconfluence in standard supplemented growth media SAGM. The cells were then washed and incubated at 37 �C with 5% CO2 in serum free/growth factor free basal medium for varying time periods. At the indicated time points, the cells were harvested, lysed and processed for isolation of RNA or protein, as described below. At confluence, the cells were harvested and total RNA extracted. Reverse transcription-polymerase chain reaction (RT-PCR) was performed on total RNA from harvested cells. Experiments were performed in triplicate, and representative data are shown.
RT-PCR
Total RNA was isolated using RNA Stat60 (Tel-Test, Friendswood, USA)
according to the manufacturer's instructions. RNA was quantified
fluorimetrically using Sybr Green II (Molecular Probes, Eugene, USA).
The isolated RNA (1 mg) was then reversely transcribed using a 20 ml volume reaction, which consisted of 10 U of Moloney murine leukemia virus reverse transcriptase (Gibco BRL, Grand Island, USA), 4 ml of 5�RT buffer (Gibco BRL), 1 ml of 10 mM deoxyribonucleotide triphosphate (dNTP; Pharmacia, Uppsala, Sweden), 2 ml of 100 mM dithiothreitol, 0.5 ml random primer pd(N)6 (Pharmacia), 0.5 ml RNasin (Promega, Madison, USA), 1 ml DPEC H2O, 10 ml total RNA. The reaction mixture was incubated for 1 h at 37 �C. Expression of CTGF mRNA transcripts was semi-quantitatively measured by PCR at 59 �C annealing temperature for 35 cycles, and compared to expression of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) housekeeping gene transcripts. The primers used for these experiments were: 5'-AAGGTGTGGCTTTAGGAGCA-3' (forward) and 5'-TTCACTTGCCAACCGCTGTC-3' (reverse) (Genset, La Jolla, USA).
The PCR products were then separated on a 2% agarose gel. The
isolated and anticipated product size was 300 bp. Sequencing analysis was
completed using the ABI PRISM377 DNA sequencer (Applied Biosystems, Foster
City, USA).
Quantitative real-time PCR
Total RNA was extracted from the bronchial epithelial cells, as
described above, and digested DNA using a DNase-treatment kit (Qiagen,
Valencia, USA). Total RNA (250-500 ng) was reversely transcribed using the Superscript RT kit
(Qiagen), and one-twentieth of the cDNA was used for real-time quantitative PCR
using the iCycler software package (Bio-Rad, Hercules, USA). The primers used
were as follows: CTGF receptor forward primer 5'-CCCTCGCGGCTTACCG-3';
CTGF receptor reverse primer 5'-GGACCAGGCAGTTGGCTCT-3'; GAPDH
forward primer 5'-GGGAAGGTGAAGGTCGGAGT-3'; GAPDH reverse
primer 5'-TCC-ACTTTACCAGAGTTAAAAGCAG-3'. The following
dual-labeled probes were obtained from BioSearch Technologies (Novato, USA):
GAPD, 5'-6-carboxy-fluorescein (6-FAM)-ACCAGGCGCCCAATACGACCAA-6-carboxy-tetramethyl-rhodamine
(6-TAMRA)-3'; CTGF; 5'-FAM-AAGACACGTTTGGCCCAGACCCAACT-black hole
quencher 2 (BHQ-2)-3'. Standards, from 10 to 0.0001 amol of the PCR
product cloned into pGEMTeasy, were run alongside the samples to generate a
standard curve. All samples and standards were analysed in triplicate. The PCR
reaction consisted of 1.5 mM Tris-HCl, 5 mM KCl, 2 mM dNTP, 200 ng of sense and
antisense primers, either 5 pmol of CTGF dual-labeled probe or 12 pmol of GAPDH
dual-labeled probe, 4 mM Mg2+ and 1 u of AmpliTaq Gold
(Applied Biosystems) in a total volume of 50 �l. The reaction conditions were
95 �C for 10 min followed by 50 cycles of 30 s at 94 �C, 30 s at 60 �C and 30 s
at 72 �C. The starting amount of cDNA in each sample was calculated using the
iCycler software package.
Antibody to CTGF protein
The commercially available CTGF polyclonal antibody developed by Torrey Pines Biomedical (La Jolla, USA) was used for immunocytochemistry. The primary antibody used was a rabbit antimouse CTGF polyclonal antibody which crossreacts with human CTGF (Torrey Pines Biomedical).
Immunocytochemistry
Bronchial epithelial cells were grown under standard conditions as
described above. Cells were rinsed briefly with phosphate-buffered saline (PBS)
and fixed in acetone-methanol (3:1) solution for 10 min at room temperature.
The primary antibody was diluted 1:100, and a control/normal rabbit serum was
also used, in a 1:100 ratio. The cells were incubated in primary antibody for
30 min, and secondary antibody fluorescein-isothiocyanate-goat F(ab) 2
antirabbit IgG (H+L; Caltag, Burlingame, USA) (1:100 dilution) for 30 min. The
cells were washed between incubations with PBS. The slides were then viewed and
photographed at final magnifications of 200� and 400�, using an
Olympus BH2 fluorescence microscope [6].
Results
RT-PCR and quantitative detection of human CTGF mRNA in HBEC
The results of RT-PCR indicated cells expressed readily detectable CTGF
transcripts. Direct sequencing of the PCR product confirmed that the generated
product was human CTGF. Quantitative real-time PCR results indicated
that, after extended starvation, bronchial epithelial cells showed a steady
decline in CTGF transcripts compared to the non-starved state, adjusted
for GAPDH. The quantitative data are expressed as the percentage or fold
change in transcripts over time [Fig. 1(A,B)].
Immunocytochemistry
The results of immunocytochemistry showed intracellular speckled cytoplasmic and perinuclear Golgi staining of the CTGF protein, as illustrated in Fig. 2(A). Fig. 2(B) shows the control cells that were incubated with rabbit serum and secondary antibody only. There was no staining in these cells. These data lend further confirmation that in human airway epithelial cells, CTGF is present and readily detectable by a variety of techniques.
Discussion
Our study was conducted to define the expression of CTGF in HBEC. These cells are metabolically active and key regulators of the inflammatory response in the lung. It is now established that in addition to lung fibroblasts, airway epithelial cells are important regulatory cells of the processing of collagen deposition and lung remodeling [7,8]. Our study demonstrates that, in vitro, normal HBEC are capable of producing CTGF, a growth factor that in turn directly regulates the deposition of collagen in the lung. We found that both proliferating non-starved and starved HBEC are able to produce CTGF. Based on our analysis, epithelial cell starvation is followed by downregulation of CTGF transcripts. One possible explanation for this observation is that cells grown under conditions of growth factor deprivation, without adding exogenous growth factors, might in turn produce factors, such as tumor necrosis factor (TNF)-a, that lead to the downregulation of CTGF expression. Another possibility is that the cells preferentially produce factors to maintain cellular proliferation, rather than CTGF, under these conditions.
The bronchial epithelial cells demonstrated both detectable CTGF
transcripts and intracellular CTGF, under the conditions studied. Our findings
have multiple implications for the ability of the bronchial epithelial cells to
directly regulate deposition of collagen and ECM, which is important in the
remodeling associated with asthma and other chronic lung diseases. Thus, intact
undamaged airway epithelial cells might participate in remodeling, as a source
of a growth factor known to directly regulate collagen deposition.
CTGF is a recently described cysteine-rich protein that regulates fibroblast proliferation and collagen deposition. CTGF is a member of a family of proteins called IGF binding proteins (IGFBPs), on the basis of its N terminal amino acid sequence homology. CTGF was provisionally named IGFBP-8 and is now designated IGFB-rP in published reports, as it has IGF-independent and unique actions as well. The regulation of CTGF expression appears to be under the control of at least one potent pro-fibrogenic cytokine secreted by a wide variety of lung cells, including epithelial cells. The CTGF promoter contains TGF-b response elements. Prostaglandin E [2] attenuates the effect of TGF-b on CTGF production. TNF-a blocks the TGF-b upregulation induced by glucocorticoids [9,10].
In clinical, CTGF expression has been identified in a wide variety of diseases involving organ fibrosis, including sarcoidosis, scleroderma, systemic sclerosis and renal fibrosis [11,12]. There are studies demonstrating that in both pediatric and adult forms of pulmonary fibrosis, CTGF could play an important regulatory role. In asthma, there are many studies indicating that a subset of patients show signs of remodeling or pulmonary fibrosis. In the lung, the interaction between epithelial cells and fibroblasts might be important in the development of lung fibrosis [13]. Based on our findings, bronchial epithelial cells could potentially directly regulate lung fibroblast proliferation and collagen deposition by means of CTGF. Further in vivo investigation should be done to study the role of CTGF in patients.
Based on published studies, CTGF has been thought to lack a role in regulating epithelial cell function, as it lacks TGF-b-like inhibitory effects on mink lung epithelial cells in vitro. In addition, the skin epidermis at sites of TGF-b administration in vivo does not demonstrate CTGF. Prior studies have demonstrated CTGF mRNA expression in the lung, although in lower abundance than the heart [14-16]. One study confirmed the presence of CTGF in alveolar type II epithelial cells from idiopathic lung fibrosis patients [15]. Our study, in contrast, demonstrates the presence of CTGF in HBEC which, unlike type II cells, are thought to be involved in the airway remodeling process.
Studies of epithelium of non-lung organs indicate there is intense CTGF staining of the epithelium within kidney collecting tubules and in olfactory, buccal, pharyngeal, esophageal and corneal organ tissue [17]. Postnatally, high levels of CTGF are also detectable in the kidney epithelium. In one study of Panc-1 epithelial pancreatic tumor cells, TGF-b-induced collagen I and CTGF production are observed [18,19]. In summary, our study demonstrates that lung epithelial cells produce CTGF. CTGF might represent a regulator of other target cells, such as lung fibroblasts and myofibroblasts.
We conclude that bronchial epithelial cells could be a novel source of CTGF, and that growth factors as well as stress might alter its expression. Bronchial epithelial cell-derived CTGF could thus directly influence the deposition of collagen in certain fibrotic lung diseases.
Acknowledgements
The author would like to thank Sandra CHAMBERS and Carol PEEBLES for their expert technical assistance, and Dr. ZURAW for review of the manuscript.
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