Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive interstitial lung disease with unknown etiology and undefined treatment modality. Fibroblasts are regarded as the major cell type that mediates the onset and progression of lung fibrosis by secreting large amounts of extracellular matrix (ECM) proteins, such as connective tissue growth factor (CTGF/CCN2). Current knowledge confers a crucial role of CCN2 in lung fibrosis. CCN5, another member of the CCN family, has been suggested to play an inhibitory role in some fibrotic diseases, such as cardiac fibrosis. However, the role of CCN5 in the process of IPF remains unknown. In the present study, using western blot analysis, we demonstrate that CCN2 is highly expressed in fibroblasts derived from IPF tissue, but is only slightly expressed in normal human lung fibroblasts. However, CCN5 was weakly expressed in all the above cells. qRT-PCR revealed that transforming growth factor (TGF)-β1 stimulation increased CCN2 expression in the IPF-derived cultures of primary human lung fibroblasts (PIFs) in a time- and concentration-dependent manner, but only slightly affected the expression of CCN5. The overexpression of CCN5 induced by the transfection of PIFs with recombinant plasmid did not affect cell viability, proliferation and apoptosis; however, it significantly suppressed the expression of CCN2, α-smooth muscle actin (α-SMA) and collagen type I. The TGF-β1-induced upregulation of the phosphorylation of Akt was reversed by CCN5 overexpression. Our results also demonstrated that adenovirus-mediated CCN5 overexpression in a mouse model of bleomycin-induced IPF significantly decreased the hydroxyproline content in the lungs, as well as TGF-β1 expression in bronchoalveolar lavage fluid. Taken together, our data demonstrate that CCN5 exerts an inhibitory effect on the fibrotic phenotypes of pulmonary fibroblasts
Idiopathic pulmonary fibrosis (IPF) is the most common form of interstitial lung disease, and generally has a poor prognosis, with an average survival of 3–5 years following diagnosis (
CCN2, which is also known as CTGF, belongs to the connective tissue growth factor/cysteine-rich 61/nephroblastoma overexpressed (CCN) family, which is composed of 6 members, including CCN1 [cysteine-rich, angiogenic inducer, 61 (CYR61)], CCN2 (CTGF), CCN3 [nephroblastoma overexpressed (NOV)], CCN4 [Wnt-induced secreted protein (WISP)-1], CCN5 (WISP-2) and CCN6 (WISP-3) (
CCN5, another member of the CCN family, also known as WISP-2, is a 29-kDa matrix protein encoded by the
In the present study, we aimed to investigate the role of CCN5 in IPF. We demonstrate that the overexpression of CCN5 in lung fibroblasts suppresses the upregulation in the expression of α-SMA and collagen induced by CCN2. Furthermore, CCN5 overexpression in lung fibroblasts decreased the expression of CCN2. The PI3K/Akt signaling pathway may be involved in the anti-fibrotic effects of CCN5. By constructing an adenovirus-mediated CCN5 expression vector, we also demonstrate that CCN5 alleviates lung fibrosis in a mouse model of bleomycin-induced IPF.
Mouse anti-human CCN2 monoclonal antibody (Ab), mouse anti-human CCN5 monoclonal Ab, mouse anti-human collagen I monoclonal Ab, mouse anti-human α-SMA monoclonal Ab, mouse anti-phospho human Akt1 (phospho S473) monoclonal Ab, mouse anti-human Akt1 monoclonal Ab, rabbit anti-phospho human JNK1 (phospho T183+Y185) polyclonal Ab, rabbit anti-human JNK1 polyclonal Ab, rabbit anti-human Smad2 polyclonal Ab, mouse anti-human α-tubulin monoclonal Ab, rabbit anti-β-actin polyclonal Ab and horseradish peroxidase (HRP)-conjugated rabbit anti-mouse immunoglobulin G (IgG) were all purchased from Abcam (Cambridge, MA, USA). Rabbit anti-phospho human Smad2 (phospho S465/S467) polyclonal Ab was purchased from Cell Signaling Technology Inc. (Beverly, MA, USA).
Human lung fibroblasts were isolated from lung biopsies following enzymatic disaggregation by collagenase and protease as previously described (
Fibroblast cell cultures and cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% heat-inactivated fetal calf serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 2 mM L-glutamine at 37°C in a 5% CO2 environment. The culture medium was changed every 2–3 days until the cells reached confluence. Cultures were used in this study after 4–8 passages. For TGF-β stimulation, fibroblast cell cultures and cell lines kept in serum-free medium were also treated with various concentrations of TGF-β1 (Sigma-Aldrich, St. Louis, MO, USA) ranging from 0.5 to 8 ng/ml for 6–72 h.
For the signaling pathway assay, the PI3K inhibitor, LY294002 (Sigma-Aldich), was added to the cultures 1 h prior to TGF-β1 stimulation. After 24 h, the cells were harvested for further assays.
Total RNA from human lung fibroblasts was extracted using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA), based on the manufacturer’s instructions. cDNA was synthesized by the reverse transcription of total RNA, using the PrimeScript RT reagent kit (Takara Bio, Inc., Dalian, China) using oligo(dT) primers, following the manufacturer’s instructions. (qRT-PCR) reactions were performed on a Bio-Rad (Hercules, CA, USA) iQ5 real-time thermal cycler using the SYBR Premix Ex Taq™ II kit (Takara Bio, Inc.). The qRT-PCR reaction system and cycling parameters were based on the Thermal Cycler Dice Real Time System. Twenty nanograms of cDNA and 0.4 μM primers were included in the 25 μl of amplification mixture. The PCR primers used were as follows: CCN2, 5′-ACCCCTGCGACCCGCACAAG-3′ (forward) and 5′-TTCACTTGCCACAAGCTGTC-3′ (reverse); CCN5, 5′-CCTCTCAAAGGTGCGTACCCA-3′ (forward) and 5′-CACGGACCATCTTCCATCAGC-3′ (reverse); β-actin, 5′-CAACTTGATGTATGAAGGCTTTGGT-3′ (forward) and 5′-ACTTTTATTGGTCTCAAGTCAGTGTACAG-3′ (reverse). These primers were all synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). The PCR procedure was as follows: polymerase activation for 30 sec at 95°C, 40 cycles of amplification each consisting of 95°C for 5 sec, 60°C for 20 sec, and 1 cycle of dissociation consisting of 95°C for 15 sec, 60°C for 30 sec and 95°C for 15 sec. All reactions were performed in triplicate, and the results were represented as relative mRNA expression data calculated according to the 2−ΔΔCT method, as previously described (
The proteins were extracted from fibroblast cells using RIPA lysis buffer (Beyotime, Nantong, China). The protein concentration in the lysates was determined using a BCA protein assay kit (Beyotime). For western blot analysis, proteins were separated on an SDS-polyacrylamide gel, transferred onto nitrocellulose membranes (Amersham Pharmacia Biotech, Piscataway, NJ,. USA) in a semi-dry trans-blot apparatus by transfer buffer, blocked with 10% defatted milk in PBS at 4°C overnight, and then incubated with the primary antibodies for 60 min. After washing with TBST buffer (0.05 mol/l Tris, 0.15 mol/l NaCl and 0.05% Tween-20), the membranes were incubated for 1 h at room temperature with HRP-conjugated secondary Ab. For detection, the ECL reagent (Boehringer Mannheim, Mannheim, Germany) was used. All experiments were performed in triplicate, and results were normalized to β-actin.
Plasmid construction and cell transfection were performed as previously described (
Cell viability assays were performed using a CellTiter-Blue cell viability assay kit (Promega Corp., Madison, WI, USA) according to the manufacturer’s instructions.
Cell proliferation was measured using the [3H]-TdR assay as previously described (
Cell apoptosis was evaluated by measuring the activity of caspase-3/7. Caspase-3/7 activity was determined using the Caspase-Glo 3/7 assay kit (Promega Corp.) following the manufacturer’s instructions. Briefly, the fibroblasts treated with the various treatments were plated on 96-well plates at a volume of 100 μl. Caspase-Glo 3/7 reagent was added to each well in a 1:1 ratio and incubated with gentle shaking for 30 min at room temperature before measuring luminescence using a microplate reader (Corona Electric Co., Ltd., Katsuta, Japan).
All recombinant adenoviruses were constructed as described in a previous study (
C57BL/6 mice (8 weeks old) were provided by the Laboratory Animal Center of Xi’an Jiaotong University. All mice were housed in specific pathogen-free conditions with free access to water. All the experiments were carried out in adherence with a protocol approved by the Institutional Animal Care and Use Committee of Xi’an Jiaotong University. The mouse model of IPF was established as previously described (
To estimate the total amount of collagen in the lungs, we measured the amount of hydroxyproline in lung tissue according to a previously described protocol (
The detection of TGF-β1 in bronchoalveolar lavage fluid (BALF) was performed as previously described (
Data are expressed as the means ± SEM from a minimum of 3 experiments. The Student’s t-test was used to determine the significance of differences in multiple comparisons. A value of P<0.05 was considered to indicate a statistically significant difference.
In light of the profibrotic function of CCN2 in pulmonary fibrosis and the putative role of CCN5 in the inhibition of fibrosis, we sought to measure the expression levels of CCN2 and CCN5 by western blot analysis in human pulmonary fibroblast cells. As expected, CCN2 was highly expressed in the PIFs and the human IPF lung fibroblastic cell lines, LL-97A and LL-29, but weakly expressed in the PNFs and the normal human lung fibroblastic cell line, LL-24 (
As we only detected a weak expression of CCN5 and its minimal alteration in response to TGF-β1, we explored the role of CCN5 by inducing the overexpression of CCN5 in PIFs. qRT-PCR and western blot analysis demonstrated that the mRNA and protein levels of CCN5, respectively were both significantly elevated by transfection with the pCEP4-Flag-CCN5 vector compared with the control group (
Fibroblast-to-myofibroblast differentiation is a critical process in the pathogenesis of a number of fibrotic diseases, including IPF. To determine whether CCN5 expression affects this process, the PIFs were transfected with pCEP4-Flag-CCN5 and then their differentiation was induced with 2 ng/ml TGF-β1 for 24 h. Western blot analysis demonstrated that the expression of α-SMA, a biomarker of myofibroblast differentiation, was markedly downregulated by CCN5 overexpression (
ECM accumulation is a prominent characteristic of the process of fibrosis. Therefore, the PIFs transfected with pCEP4-Flag-CCN5 plasmid were stimulated with 2 ng/ml TGF-β1 for 24 h, and the alteration in the expression of collagen type I was examined by western blot analysis. Compared with the control group, collagen type I expression was markedly reduced by CCN5 overexpression (
It has been previously suggested that the Smads, and the PI3K/Akt and JNK pathway are involved in TGF-β1-induced fibrosis (
To determine the inhibitory effects of CCN5 overexpression on lung fibrosis
Although CCN5 has been suggested to play an anti-fibrotic role in some fibrotic diseases, the role of CCN5 in IPF remains unknown. In the present study, we demonstrate that CCN5 overexpression inhibits the fibrotic phenotype
IPF is a specific form of chronic fibrotic interstitial pneumonia with a poor prognosis. The concept of IPF pathogenesis has progressed from chronic inflammation to aberrant wounding healing and, even more recently, to the current paradigms of a multifactorial and heterogeneous disease process (
Previous studies have revealed that the upregulation of collagen and TGF-β are markedly exacerbated in CCN2 transgenic mice, but are inhibited in CCN5 transgenic mice (
Apart from the Smad-dependent pathway, Smad-independent pathways, such as PI3K/Akt have also been suggested to be involved in TGF-β signaling (
We then investigated the effects of CCN5 in mcie with bleomycin-induced IPF using recombinant adenoviral vectors expressing CCN5. CCN5 overexpression in the lungs abrogated the bleomycin-induced upregulation of CCN2 and collagen in the tissue. These
In conclusion, our results demonstrate that CCN5 exerts an inhibitory effect on the fibrotic phenotypes in lung fibroblasts derived from patients with IPF and
This study was supported by a grant from the Technology Plan Projects of Shaanxi province (no. 2011k14-10-01). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
CCN2 and CCN5 expression patterns in pulmonary fibroblasts. (A) CCN2 and CCN5 expression patterns were detected by western blot analysis in primary lung fibroblasts derived from patients with idiopathic pulmonary fibrosis (PIFs), the human IPF fibrobastic cell lines, LL-97A and LL-29, and primary human normal lung fibroblasts (PNFs) and normal human fibroblastic cell line, LL-24. (B) PIFs was pre-incubated with TGF-β1 (range, 0–8 ng/ml) for 24 h. CCN2 and CCN5 expression was detected by qRT-PCR. (C) PIFs were pre-incubated with TGF-β1 (2 ng/ml) for up to 72 h. CCN2 and CCN5 expression was detected byqRT-PCR. Cells were pre-treated with TGF-β1 (2 ng/ml) for 12 h. (D) CCN2 and (E) CCN5 levels were determined by qRT-PCR. The mRNA expression levels was normalized based on β-actin levels. Relative mRNA expression levels were the ratio of the normalized mRNA expression levels in the treatment group to those in the control group. Data are expressed as the means ± SEM of 3 independent experiments. P<0.05 was considered to indicate a statistically significant difference.
Effects of CCN5 overexpression on the viability, proliferation and apoptosis of pulmonary fibroblasts. Primary lung fibroblasts derived from patients with idiopathic pulmonary fibrosis (PIFs) were transfected with the pCEP4-Flag-CCN5 recombinant plasmid. The expression of CCN2 and CCN5 in the control group, vector group and pCEP4-Flag-CCN5-transfected group was determined by (A) qRT-PCR and (B) western blot analysis. (C) Cell viability was determined by a CellTiter-Blue Cell Viability assay kit. (D) Cell proliferation was determined by [3H]-thymidine incorporation assay. Cells were left unstimulated or pre-incubated with TGF-β1 for 24 h. Data are expressed as the percentage incorporation of [3H]-thymidine compared with the control group. CPM, counts per minute. (E) Cell apoptosis was determined by measuring the activity of caspase-3/7. Cells were left unstimulated or pre-incubated with TGF-β1 for 24 h. Data are expressed as means ± SEM of 3 independent experiments. P<0.05 was considered to indicate a statistically significant difference.
Effect of CCN5 overexpression on the expression of α-smooth muscle actin (α-SMA) and collagen type I. Primary lung fibroblasts derived from patients with idiopathic pulmonary fibrosis (PIFs) were transfected with the pCEP4-Flag-CCN5 recombinant plasmid. Following stimulation with 2 ng/ml TGF-β1 for 24 h, the cells were examined for (A) α-SMA and (B) collagen I expression assay by western blot analysis. Data are expressed as the means ± SEM of 3 independent experiments. P<0.05 was considered to indicate a statistically significant difference.
(CCN5 overexpression attenuates the phosphorylation of Akt. Primary lung fibroblasts derived from patients with idiopathic pulmonary fibrosis (PIFs) were either left untreated, stimulated with TGF-β1, pre-treated with PI3K inhibitor, LY294002, or transfected with pCEP4-Flag-CCN5 prior to TGF-β1 stimulation. (A) Western blot analysis was used to determine the levels of phosphorylated Smad2 (p-Smad2) and total Smad2 (t-Smad2), JNK and Akt. The intensity of the bands probed with (B) anti-p-Smad2, (C) anti-p-JNK and (D) anti-p-Akt antibodies was quantified and normalized to the corresponding total protein. Data are expressed as the means ± SEM of 3 independent experiments. *P<0.05 vs. the control group and #P<0.05 vs. the TGF-β1 treated group.
Effects CCN5 overexpression on fibrotic response in lungs of mice with bleomycin-induced lung fibrosis. Mice were either left untreated, pre-treated with normal saline, pre-treated with Ad-GFP or pre-treated with Ad-CCN5 prior to the administration of bleomycin. (A) Mice were sacrificed on day 14 and samples of the lung tissue were used for protein extraction. CCN2 and CCN5 expression levels were determined by western blot analysis. (B) Hydroxyproline content at 14 days in the lungs of mice with bleomycin-induced lung fibrosis was determined using a hydroxyproline assay. (C) Bronchoalveolar lavage fluid (BALF) was collected on day 14, and levels of TGF-β1 in the BALF were measured with an ELISA kit using a standardized procedure. Data are expressed as the means ± SEM. *P<0.05 vs. the normal untreated group and #P<0.05 vs. the group pre-treated with normal saline and administered bleomycin (n=10).