Smad3 mediates TGF-β1-induced collagen gel contraction by human lung fibroblasts

doi:10.1016/j.bbrc.2005.10.209

Biochemical and Biophysical Research Communications

Volume 339, Issue 1, 6 January 2006, Pages 290-295

https://doi.org/10.1016/j.bbrc.2005.10.209 Get rights and content

Abstract

Transforming growth factor-β₁ (TGF-β₁) is a key mediator in tissue repair and fibrosis. Using small interference RNA (siRNA), the role of Smad2 and Smad3 in TGF-β stimulation of human lung fibroblast contraction of collagenous matrix and induction of α-SMA and the role of α-SMA in contraction were assessed. HFL-1 cells were transfected with Smad2, Smad3 or control-siRNA, and cultured in floating Type I collagen gels ±−TGF-β₁. TGF-β₁ augmented gel contraction in Smad2-siRNA- and control-siRNA-treated cells, but had no effect in Smad3-siRNA-treated cells. Similarly, TGF-β₁ upregulated α-SMA in Smad2-siRNA- and control-siRNA-treated cells, but had no effect on Smad3-siRNA-treated cells. α-SMA-siRNA-treated cells did not contact the collagen gels with or without TGF-β₁, suggesting α-SMA is required for gel contraction. Thus, Smad3 mediates TGF-β₁-induced contraction and α-SMA induction in human lung fibroblasts. Smad3, therefore, could be a target for blocking contraction of human fibrotic tissue induced by TGF-β₁.

Section snippets

Materials and methods

Cell culture. Human fetal lung fibroblasts (HFL-1) were obtained from the American Type Culture Collection (Rockville, MD). The cells were cultured in 100-mm tissue culture dishes (Falcon; Becton–Dickinson Labware, Lincoln Park, NJ) in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal calf serum (FCS), 50 U/ml penicillin G sodium, 50 μg/ml streptomycin sulfate (penicillin–streptomycin; Invitrogen, Life Technologies, Grand Island, NY), and 1 μg/ml amphotericin B (pharma-Tek,

Smad3 mediates the TGF-β₁-induced collagen gel contraction by human lung fibroblasts

Inhibition of HFL-1 Smad2 and Smad3 protein expression after treating with the siRNA used has been assessed by Western blot and previously reported [7]. To evaluate transcriptional activation of Smad3, we used reporter constructs specific for Smad3 (CAGA12) [10], [11]. As expected, transfection with Smad3-siRNA resulted in almost 100% reduction in TGF-β₁-stimulated CAGA12 reporter activity. This extends the previous results obtained by Western blot and demonstrates inhibition of TGF-β-induced

Discussion

Fibroblasts cultured in a three-dimensional collagen matrix attach to the collagen fibers and generate mechanical tension [5], [6]. If maintained in floating culture, fibroblasts will cause the surrounding matrix to contract [12]. This in vitro effect is believed to be a model of the contraction that characterizes both wound healing and the development of fibrosis. Transforming growth factor beta (TGF-β) stimulates the process [13]. The current study demonstrates that, in human fibroblasts, the

Acknowledgments

We appreciate the helpful discussions with Dr. A. Roberts, National Institutes of Health (NIH), and the secretarial support of Ms. Lillian Richards. This work was supported by NIH Grant 5 R01 HL64088-04.

References (21)

G.R. Grotendorst
Connective tissue growth factor: a mediator of TGF-beta action on fibroblasts
Cytokine Growth Factor Rev.
(1997)
T. Kobayashi et al.
Smad3 mediates TGF-beta1 induction of VEGF production in lung fibroblasts
Biochem. Biophys. Res. Commun.
(2005)
A.K. Kamaraju et al.
Role of Rho/ROCK and p38 MAP kinase pathways in transforming growth factor-beta-mediated Smad-dependent growth inhibition of human breast carcinoma cells in vivo
J. Biol. Chem.
(2005)
F. Grinnell et al.
Transforming growth factor beta stimulates fibroblast-collagen matrix contraction by different mechanisms in mechanically loaded and unloaded matrices
Exp. Cell Res.
(2002)
M.B. Vaughan et al.
Transforming growth factor-beta1 promotes the morphological and functional differentiation of the myofibroblast
Exp. Cell Res.
(2000)
W.R. Roche et al.
Subepithelial fibrosis in the bronchi of asthmatics
Lancet
(1989)
A. Leask et al.
TGF-beta signaling and the fibrotic response
FASEB J.
(2004)
G.C. Blobe et al.
Role of transforming growth factor beta in human disease
N. Engl. J. Med.
(2000)
X. Liu et al.
Smad3 mediates the TGF-beta induced contraction of type 1 collagen gels by mouse embryo fibroblasts
Cell Motil. Cytoskeleton
(2003)
F. Grinnell
Fibroblasts, myofibroblasts and wound contraction
J. Cell Biol.
(1994)

There are more references available in the full text version of this article.

Cited by (63)

Suspended particulate matter promotes epithelial-to-mesenchymal transition in alveolar epithelial cells via TGF-β1-mediated ROS/IL-8/SMAD3 axis
2024, Journal of Environmental Sciences (China)
Epidemiological evidence presents that dust storms are related to respiratory diseases, such as pulmonary fibrosis (PF). However, the precise underlying mechanisms of SPM-elicited adverse effects still need to be investigated. Epithelial-mesenchymal transition (EMT) process is a characteristic of PF. We discussed whether suspended particulate matter (SPM) is involved in EMT induction via transforming growth factor-β1 (TGF-β1). In this study, a detailed elemental analysis (55 elements), particle size, and morphology were determined. To investigate the toxicity of SPM, an MTT test was performed to detect cell viability. Next, A549 cells were exposed to selected concentrations of SPM (20 and 40 µg/mL) for single and repeated exposures. The DCFH-DA assay showed that exposure to SPM could produce reactive oxygen species (ROS). The ELISA assay demonstrated increased levels of interleukin-8 (IL-8) and TGF-β1 in the supernatant. Western blot was used to detect the expression of proteins associated with EMT and the SMAD3-dependent pathway. Results of western blot demonstrated that E-cadherin was reduced, whereas p-SMAD3, vimentin, and α-smooth muscle actin were elevated. Our findings indicated that SPM triggered EMT by induction of oxidative stress, inflammation, and the TGF-β1/SMAD3 pathway activation.
Intermittent mild cold stimulation alleviates cold stress-induced pulmonary fibrosis by inhibiting the TGF-β1/Smad signaling pathway in broilers
2024, Poultry Science
To investigate the potential protective effect of intermittent cold stimulation on lung tissues of broilers exposed to acute cold stress (ACS). A total of 384 one-day-old broilers were assigned to 4 experimental groups with 6 replicates of 16 birds each: control (CON) and ACS groups were reared at normal feeding temperature from d 1 to 42; cold treatment groups (CS3+ACS and CS9+ACS) were reared, respectively, at 3°C or 9°C for 5 h on alternate days below the CON group from d 15 to 35. Animals in CS3+ACS, CS9+ACS, and ACS groups were exposed at 10°C for 24 h on d 43. Subsequently, lung tissues were collected to perform histopathological examination and measurement of relevant indexes. The results showed that lung tissues in CS9+ACS and ACS groups exhibited increased inflammatory cell infiltrates and collagen deposition compared to the CON group, while this pathological phenomenon was less pronounced in the CS3+ACS group. Compared to CON group, H₂O₂ and MDA contents were increased, and the activities of antioxidant enzymes (CAT, SOD, GPx, T-AOC) were reduced in CS9+ACS and ACS group (P < 0.05); mRNA and protein levels of inhibitor of NF-κB, Smad7, matrix metallopeptidase (MMP)-2, MMP9, and antioxidant-related genes were downregulated, whereas mRNA and protein levels of genes related to NF-κB/NLRP3 pathway-regulated inflammation and TGF-β1/Smad pathway-regulated fibrosis were upregulated in cold-stressed broilers (P < 0.05). mRNA levels of heme oxygenase-1, NAD(P)H quinone oxidoreductase-1, and MMP9 were increased in CS3+ACS group (P < 0.05). Moreover, the expression of most antioxidant-related genes was increased, and that of inflammation- and fibrosis-related genes was reduced in CS3+ACS group (P < 0.05). Therefore, cold stress caused oxidative stress and inflammation, leading to pulmonary fibrosis in broilers, whereas intermittent mild cold stimulation at 3°C below normal rearing temperature alleviated fibrosis by inhibiting the TGF-β1/Smad pathway modulated by the Nrf2/HO-1 and NF-κB/NLRP3 signaling pathway. This study suggests that intermittent mild cold stimulation can be a potential strategy to reduce ACS-induced lung damage in broilers.
VX-765 attenuates silica-induced lung inflammatory injury and fibrosis by modulating alveolar macrophages pyroptosis in mice
2023, Ecotoxicology and Environmental Safety
Silicosis is a diffuse fibrotic lung disease in which excessive inflammatory responses are triggered by silica exposure. Pyroptosis, a pro-inflammatory mode of programmed cell death, is mediated by gasdermin and may play a pivotal role in the development of silicosis. The caspase-1 inhibitor, VX-765, was used in vivo and in vitro to investigate the effects of silica-induced early inflammatory injury and later lung fibrosis. Our findings show that VX-765 reduces inflammatory lung injury by inhibiting silica-induced pyroptosis of alveolar macrophages in a silicosis mouse model. VX-765 limits the infiltration of inflammatory M1 alveolar macrophages, decreasing expression of inflammatory cytokines, including IL-1β, TNF-α, IL-6, CCL2, and CCL3, and down-regulating endogenous DAMPs and inflammatory immune-related cell pattern recognition receptors TLR4 and NLRP3. Furthermore, VX-765 alleviates fibrosis by down-regulating α-smooth muscle actin (α-SMA), collagen, and fibronectin. In this study, we illustrate that Alveolar macrophages pyroptosis occur in the early stages of silicosis, and VX-765 can alleviate the development of silicosis by inhibiting the pyroptosis signaling pathway. These results may provide new insight into the prevention and treatment of early-stage silicosis.
Application of glucagon-like peptide-1 receptor antagonists in fibrotic diseases
2022, Biomedicine and Pharmacotherapy
Fibrosis can occur in various organs, leading to structural destruction, dysfunction, and even organ failure. Hence, organ fibrosis is being actively researched worldwide. Glucagon-like peptide-1 (GLP-1), a naturally occurring hormone, binds to a G-protein-coupled receptor widely distributed in the pancreas, kidney, lung, heart, gastrointestinal tract, and other organs. Synthetic GLP-1 analogs can be used as GLP-1 receptor agonists (GLP-1RAs) for treating diabetes mellitus. In recent years, GLP-1RAs have also been found to exert anti-inflammatory, antioxidant, and cardiovascular protective effects. GLP-1RAs have also been shown to inhibit fibrosis of solid organs, such as the lung, heart, liver, and kidney. In this review, we discuss the advancements in research on the role of GLP-1RAs in the fibrosis of the heart, lung, liver, kidney, and other organs to obtain new clues for treating organ fibrosis.
Tanshinone IIA attenuates silica-induced pulmonary fibrosis via Nrf2-mediated inhibition of EMT and TGF-β1/Smad signaling
2020, Chemico-Biological Interactions
Silicosis is an occupational pulmonary fibrosis that is caused by inhalation of silica (SiO₂), and there are no effective drugs to treat this disease. Tanshinone IIA (Tan IIA), a natural product, has been reported to possess antioxidant and anti-fibrotic properties in various diseases. The purpose of the current study was to examine Tan IIA's protective effects against silica-induced pulmonary fibrosis and to explore the underlying mechanisms. We found that in vivo treatment with Tan IIA significantly relieved silica-induced lung fibrosis in a silicosis rat model by histological and immunohistochemical analyses. Further, in vitro mechanistic investigations, mainly using western blot and immunofluorescence analyses, revealed that Tan IIA administration markedly inhibited the silica-induced epithelial-mesenchymal transition (EMT) and transforming growth factor-β1 (TGF-β1)/Smad signaling pathway and also reduced silica-induced oxidative stress and activated the nuclear factor erythroid 2-related factor-2 (Nrf2) signaling pathway in A549 and human bronchial epithelial (HBE) cells. Furthermore, through transfection with siRNA, we demonstrate that Nrf2 activation partially mediates the suppression effects of Tan IIA on EMT and TGF-β1/Smad signaling pathway activation induced by silica exposure, thus mediating the anti-fibrotic effects of Tan IIA against silica-induced pulmonary fibrosis. In our study, Tan IIA has been identified as a possible anti-oxidative and anti-fibrotic drug for silicosis.
Tanshinone IIA attenuates silica-induced pulmonary fibrosis via inhibition of TGF-β1-Smad signaling pathway
2020, Biomedicine and Pharmacotherapy
Transforming growth factor-β 1 (TGF-β1) is a key mediator in fibrogenesis, and is upregulated and activated in fibrotic diseases. The exact role of TGF-β1-Smad signaling in the progression of silicosis fibrosis is yet to be conclusively determined. Using a Wistar rat silicosis model, we examined whether tanshinone IIA (Tan IIA) could meliorate silicosis fibrosis. The pulmonary fibroblasts of rats from the normal control group and silicosis-induced model group were extracted and examined so as to further explore the disruption of TGF-β1-Smad signaling pathway in silicosis pathogenesis and the intervention of Tan IIA in this pathway. Using RT-PCR, immunohistochemical staining, and immunofluorescence analysis, we determined that Tan IIA could ameliorate silicosis fibrosis, downregulate collagen I, collagen III, and α-SMA expression both, in vivo and in vitro. In silicosis fibroblasts, TGF-β1 induced phosphorylation of Smad2, Smad3, and negative feedback Smad7 inhibition in a dose dependent manner, and the phosphorylation of Smad3 persisted when the upstream signal was blocked. Tan IIA treatment effectively inhibited the TGF-β1-induced phosphorylation of Smads, especially the persistent phosphorylation of Smad3 in the nucleus, and upregulated the expression of Smad7 in silicosis fibroblasts, leading to a reduction in ECM deposition. Our findings indicate that dysregulation of the TGF-β1-Smad signaling pathway may play an important role in the pathological process of silicosis. Tan IIA thus ameliorates silicosis fibrosis partially by suppressing activation of TGF-β1-Smad signaling pathway, which may turn out to be a potential therapeutic approach to prevent silicosis fibrosis.

View all citing articles on Scopus

View full text

Smad3 mediates TGF-β1-induced collagen gel contraction by human lung fibroblasts

Abstract

Section snippets

Materials and methods

Smad3 mediates the TGF-β1-induced collagen gel contraction by human lung fibroblasts

Discussion

Acknowledgments

Cytokine Growth Factor Rev.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Exp. Cell Res.

Exp. Cell Res.

Lancet

TGF-beta signaling and the fibrotic response

FASEB J.

Role of transforming growth factor beta in human disease

N. Engl. J. Med.

Smad3 mediates the TGF-beta induced contraction of type 1 collagen gels by mouse embryo fibroblasts

Cell Motil. Cytoskeleton

Fibroblasts, myofibroblasts and wound contraction

J. Cell Biol.

Smad3 mediates TGF-β₁-induced collagen gel contraction by human lung fibroblasts

Smad3 mediates the TGF-β₁-induced collagen gel contraction by human lung fibroblasts