Collagen Synthesis In Lung Fibroblasts Research Articles

Piceatannol-mediated JAK2/STAT3 signaling pathway inhibition contributes to the alleviation of oxidative injury and collagen synthesis during pulmonary fibrosis

Pulmonary fibrosis (PF) is characterized by oxidative injury and excessive collagen synthesis in lung fibroblasts, causing impaired pulmonary function and chronic lung injury. Piceatannol, a dietary polyphenol, possesses vital pharmacological effects in metabolic disorders, cancers, cardiovascular disease and infectious disease; however, its role in PF is still not completely elucidated. Mice (8 to 10 weeks old) were administered bleomycin (BLM) intratracheally (2 U/kg) to establish an in vivo PF model. Murine primary lung fibroblasts were isolated and stimulated with TGF-β (10 ng/mL) for 48 h to induce its activation. Meanwhile, mice or primary lung fibroblasts were treated with different doses of piceatannol to observe its protective roles. Pulmonary function and arterial blood gas were detected to assess pulmonary physiological status. Collagen deposition and the mRNA levels of profibrotic genes were determined by H&E staining and RT-PCR. Meanwhile, the protein and mRNA markers, as well as end-product of oxidative stress were detected in vivo and in vitro. The results showed that pulmonary function was significantly impaired in BLM-induced mice, accompanied by elevated oxidative stress and excessive collagen synthesis. Piceatannol significantly improved pulmonary function and decreased oxidative injury as well as collagen synthesis in mice with PF. Mechanically, piceatannol treatment significantly inhibited the activation of JAK2/STAT3 signaling pathway in BLM-induced mice and TGF-β-induced lung fibroblasts. Additional findings also demonstrated that coumermycin A1 (C-A1), an agonist of JAK2, could abolish the effects of piceatannol on TGF-β-induced lung fibroblasts and reactivated the phosphorylation STAT3. Taken together, our study demonstrated that piceatannol could protect against oxidative injury and collagen synthesis during PF in a JAK2/STAT3 signaling pathway-dependent manner.

Metformin prevents PFKFB3-related aerobic glycolysis from enhancing collagen synthesis in lung fibroblasts by regulating AMPK/mTOR pathway.

Aerobic glycolysis has been shown to contribute to the abnormal activation of lung fibroblasts with excessive collagen deposition in lipopolysaccharide (LPS)-induced pulmonary fibrosis. Targeting aerobic glycolysis in lung fibroblasts might therefore be considered as a promising therapeutic approach for LPS-induced pulmonary fibrosis. In the present study, the aim was to investigate whether metformin, a widely used agent for treating type 2 diabetes, could alleviate LPS-induced lung fibroblast collagen synthesis and its potential underlying mechanisms. Different concentrations of metformin were used to treat the human lung fibroblast MRC-5 cells after LPS challenge. Indicators of aerobic glycolysis in MRC-5 cells were detected by measuring glucose consumption and lactate levels in culture medium in addition to lactate dehydrogenase activity in cellular lysates. The glucose consumption, lactate levels and the lactate dehydrogenase activity were measured respectively using colorimetric/fluorometric and ELISA kits. The effects of metformin in AMP-activated protein kinase (AMPK) activation was assessed by mitochondrial complex I activity kits. Collagen I, α-smooth muscle actin (α-SMA) and collagen III were used as markers of collagen synthesis, which was measured using western blotting, whereas phosphorylated (p-) AMPK, AMPK, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) and mTOR were detected by western blotting. Metformin significantly decreased mitochondrial complex I activity and upregulated the expression of p-AMPK/AMPK protein in a concentration-dependent manner. Furthermore, the aerobic glycolysis mediated by PFKFB3 and collagen synthesis in LPS-treated MRC-5 cells was gradually inhibited with increasing concentrations of metformin. However, this inhibitory role of metformin on PFKFB3-meditaed aerobic glycolysis and collagen synthesis was prevented by treatments with 3BDO and compound C, which are specific mTOR activator and AMPK inhibitor, respectively. Taken together, the findings from this study suggested that metformin may prevent PFKFB3-associated aerobic glycolysis from enhancing collagen synthesis in lung fibroblasts via regulating the AMPK/mTOR pathway.

Mir-21 Mediates the Inhibitory Effect of Ang (1\u20137) on AngII-induced NLRP3 Inflammasome Activation by Targeting Spry1 in lung fibroblasts

MicroRNA-21 (mir-21) induced by angiotensin II (AngII) plays a vital role in the development of pulmonary fibrosis, and the NLRP3 inflammasome is known to be involved in fibrogenesis. However, whether there is a link between mir-21 and the NLRP3 inflammasome in pulmonary fibrosis is unknown. Angiotensin-converting enzyme 2/angiotensin(1–7) [ACE2/Ang(1–7)] has been shown to attenuate AngII-induced pulmonary fibrosis, but it is not clear whether ACE2/Ang(1–7) protects against pulmonary fibrosis by inhibiting AngII-induced mir-21 expression. This study’s aim was to investigate whether mir-21 activates the NLRP3 inflammasome and mediates the different effects of AngII and ACE2/Ang(1–7) on lung fibroblast apoptosis and collagen synthesis. In vivo, AngII exacerbated bleomycin (BLM)-induced lung fibrosis in rats, and elevated mir-21 and the NLRP3 inflammasome. In contrast, ACE2/Ang(1–7) attenuated BLM-induced lung fibrosis, and decreased mir-21 and the NLRP3 inflammasome. In vitro, AngII activated the NLRP3 inflammasome by up-regulating mir-21, and ACE2/Ang(1–7) inhibited NLRP3 inflammasome activation by down-regulating AngII-induced mir-21. Over-expression of mir-21 activated the NLRP3 inflammasome via the ERK/NF-κB pathway by targeting Spry1, resulting in apoptosis resistance and collagen synthesis in lung fibroblasts. These results indicate that mir-21 mediates the inhibitory effect of ACE2/Ang(1–7) on AngII-induced activation of the NLRP3 inflammasome by targeting Spry1 in lung fibroblasts.

Induction of MiR-21 by Stereotactic Body Radiotherapy Contributes to the Pulmonary Fibrotic Response

Radiation-induced lung fibrosis, the most serious effect of lung cancer radiotherapy on normal tissue, remains a major technical obstacle to the broader application of radiotherapy to patients with lung cancer. This study describes the use of an image-guided irradiation system in mice mimicking stereotactic body radiotherapy (SBRT) to examine the molecular features of chronic fibrotic response after radiation injury. MicroRNA (miR) array analysis of injured pulmonary tissue identified a set of miRs whose expression was significantly increased in damaged lung tissue. In particular, miR-21 expression was increased at the radiation injury site, concurrent with collagen deposition. Although the inhibition of miR-21 by its specific inhibitor anti-miR-21 only marginally affected endothelial-mesenchymal transition (EndMT) in lung endothelial cells, this inhibition significantly reduced collagen synthesis in lung fibroblasts. Furthermore, ectopic expression of miR-21 was sufficient to promote a fibrotic response in lung fibroblasts, enhancing Smad2 phosphorylation concurrent with Smad7 downregulation. These findings indicate that the induction of miR-21 expression is responsible for fibrotic responses observed in mesenchymal cells at the injury site through the potentiation of TGF-β signaling. Local targeting of miR-21 at the injured area could have potential therapeutic utility in mitigating radiation-induced lung fibrosis.

IL-27 attenuates the TGF-β1-induced proliferation, differentiation and collagen synthesis in lung fibroblasts

AimsPulmonary fibrosis is a type of chronic lung disease and has characteristics that progress quickly, has a high fatality rate and a poor therapeutic effect. Our previous research showed that interleukin-27(IL-27) potentially attenuates BLM-induced pulmonary fibrosis, but the function of IL-27 in lung fibroblasts (LFs) differentiation pulmonary fibrosis is yet to be known. Main methodsHere we investigated the effect of IL-27 on the proliferation, differentiation and collagen synthesis of lung fibroblasts induced by transforming growth factor-β1 (TGF-β1)using MTT, bromodeoxyuridine(BrdU) staining, real-time quantitative PCR(qPCR), Western blot, cell cycle FACS assay and immunofluorescence. We also examined the expression of the JAK/STAT and TGF-β1/Smad signaling pathway of IL-27 in lung fibroblasts. Key findingsTGF-β1 treated lung fibroblasts showed significantly increased proliferation, differentiation and collagen synthesis as well as overactivated JAK/STAT and TGF-β1/Smad signaling. However, the presence of IL-27 weakened these effects and obviously inactivated the JAK/STAT and TGF-β1/Smad signaling pathways. SignificanceOur results indicate that IL-27 may play an anti-fibrotic role in the development, differentiation and collagen synthesis in lung fibroblasts. These data also may provide a target gene therapy method in treating pulmonary fibrosis.

Prostaglandin D2 Inhibits Collagen Secretion From Lung Fibroblasts by Activating the DP Receptor

Lung fibroblasts are responsible for collagen secretion during normal tissue repair and the development of fibrosis. Many other prostaglandins have been reported to regulate collagen synthesis in lung fibroblasts, but the role of prostaglandin D2 (PGD2) is unknown. In this study, we investigated the effect of PGD2 on type I collagen secretion in human lung fibroblasts. Pretreatment with PGD2 (0.1 - 10 μM, 1 h) significantly attenuated type I collagen secretion to the cell supernatant induced by transforming growth factor-β (TGF-β). Although an agonist on chemoattractant receptorhomologous molecule expressed on Th2 cells (CRTH2) did not have any effect, the prostanoid DP-receptor agonist BW245C (0.01 - 1 μM) suppressed TGF-β-induced collagen secretion. PGD2 and BW245C significantly increased intracellular cAMP level. One-hour pretreatment with forskolin (0.1 - 10 μM), dibutyryl-cAMP (0.01 - 1 mM), and the protein kinase A (PKA)-activator N(6)-phenyl-cyclic AMP (100 μM) significantly reduced TGF-β-induced collagen secretion, while exchange protein activated by cAMP (Epac) activator 8-bromo-2'-O-methyladenosine-3',5'-cyclic AMP (10 μM) did not affect collagen deposition. These results suggest that PGD2 inhibits TGF-β-induced collagen secretion via intracellular cAMP accumulation through activating DP receptor.

Myocardin-related Transcription Factor-A Complexes Activate Type I Collagen Expression in Lung Fibroblasts

Pulmonary fibrosis is characterized by the excessive deposition of a collagen-rich extracellular matrix. The accumulation of collagen within the lung interstitium leads to impaired respiratory function. Furthermore, smooth muscle actin-positive myofibroblasts within the fibrotic lung contribute to disease progression. Because collagen and smooth muscle cell α-actin are coordinately expressed in the setting of fibrosis, the hypothesis was tested that specific transcriptional regulators of the myocardin family might also regulate collagen gene expression in myofibroblasts. Myocardin-related transcription factors (MRTFs), through their interaction with the serum-response factor (SRF) on CArG box regulatory elements (CC(A/T)6GG), are important regulators of myofibroblast differentiation. MRTF-A transactivated type I collagen gene reporters as much as 100-fold in lung myofibroblasts. Loss of functional MRTF-A using either a dominant negative MRTF-A isoform, shRNA targeting MRTF-A, or genetic deletion of MRTF-A in lung fibroblasts significantly disrupted type I collagen synthesis relative to controls. Analysis of the COL1A2 proximal promoter revealed a noncanonical CArG box (CCAAACTTGG), flanked by several Sp1 sites important for MRTF-A activation. Chromatin immunoprecipitation experiments confirmed the co-localization of MRTF-A, SRF, and Sp1 bound to the same region of the COL1A2 promoter. Mutagenesis of either the noncanonical CArG box or the Sp1 sites significantly disrupted MRTF-A activation of COL1A2. Together, our findings show that MRTF-A is an important regulator of collagen synthesis in lung fibroblasts and exhibits a dependence on both SRF and Sp1 function to enhance collagen expression.

Evidence for the involvement of TGF-β and PDGF in the regulation of prolyl 4-hydroxylase and lysyloxidase in cultured rat lung fibroblasts

Lung fibrosis is the end-point of numerous lung disorders induced by a pneumonia or by a variety of different noxes, one of which is the cytostatic drug bleomycin (BLM). Fibrosis is characterized by excessive extracellular matrix accumulation. Macrophage-fibroblast interactions are suggested to play an important role in the development of this disease. The present study was addressed to investigate one possible pathway of this interaction, the influence of soluble mediators produced by BLM-stimulated macrophages on lung fibroblast collagen synthesis and modification. Conditioned media (CM) of BLM-exposed macrophages of the cell line NR8383 submitted to rat lung fibroblast cultures increased the activity of prolyl 4-hydroxylase (P4H) in fibroblasts in a dose dependent manner. CM of stimulated macrophages increased the collagen concentration in fibroblast culture supernatant. The level of mRNAs specific for the alpha-subunit of P4H and that for alpha1(I) collagen were found to be increased by about two-fold, that for lysyloxidase (LO) by about 2.5-fold in fibroblasts cultured in CM of stimulated macrophages. Pre-incubation of CM of BLM-exposed macrophages with neutralizing antibodies against TGF-beta or against PDGF resulted in a partial reversal of the increasing effect of the CM on P4H- and LO-activities in fibroblasts. Both growth factors, TGF-beta and PDGF, added to fibroblast cultures led to significant increases of P4H activity in the treated cells. We conclude that TGF-beta and PDGF produced by stimulated macrophages are involved in the regulation of the expression of alpha1(I) collagen, of P4H-alpha-subunit and LO in lung fibroblasts. The results indicate that this is not a direct effect but involves the action of a so far unidentified mediator responsible for autocrine stimulation of collagen production.

Rational basis for oligodeoxynucleotides to inhibit collagen synthesis in lung fibroblasts and primary fibroblasts from liver granulomas of Schistosoma mansoni-infected mice

Hepatocellular carcinoma is associated with liver fibrosis. Murine schistosomiasis infection offers a model to study hepatic fibrogenesis. Single-stranded phosphorothiate oligodeoxynucleotides containing the TGF-β regulatory element have been shown to regulate the transcription of this gene and effectively inhibit collagen synthesis in primary fibroblasts isolated from schistosomiasis-induced hepatic granulomas. While the single-stranded oligos did not decrease collagen and non-collagen protein synthesis below control levels, their double-stranded modified and unmodified counterparts did. Competitive cold oligodeoxynucleotide gel mobility shift analysis using control fibroblast nuclear extract demonstrated that the single-stranded oligos diminished binding of the TGF-β activator protein to the TGF-β regulatory element while the double-stranded oligos totally inhibited this binding. TGF-β element containing single-stranded phosphorothioate oligodeoxynucleotides and their double-stranded counterparts may be successful therapeutic agents to inhibit hepatic fibrogenesis and associated hepatocellular carcinoma.

Perspective article: transforming growth factor-beta: crossroad of glucocorticoid and bleomycin regulation of collagen synthesis in lung fibroblasts.

Fibrosis is a consequence of injury which is characterized by accumulation of excess collagen and other extracellular matrix components, resulting in the destruction of normal tissue architecture and function. Transforming growth factor-beta, a potent wound healing agent, has also been shown to be an agent that can produce fibrosis because it is a potent stimulator of collagen synthesis. Both glucocorticoids and bleomycin have recently been shown to affect collagen synthesis in opposite directions, by utilizing a common pathway of involving transforming growth factor-beta activator protein binding to the transforming growth factor-beta element. This article presents a mechanistic overview of collagen synthesis regulation by glucocorticoids and bleomycin through the transforming growth factor-beta pathway.

Transforming growth factor-beta is generated in the course of hypersensitivity pneumonitis: contribution to collagen synthesis.

Mice of the C57BL/6 strain were instilled with optimal doses (150 micrograms/day for 3 days/wk) of the thermophilic actinomycete Faeni rectivirgula (also known as Saccharopolyspora rectivirgula or Micropolyspora faeni) to induce a hypersensitivity pneumonitis inflammation that mimics the human disease affecting certain occupational groups. This mouse model was characterized by a very significant alveolitis (3-fold increase in bronchoalveolar lavage [BAL] cell number at 48 h and a 10-fold increase at 3 wk). Also, total lung transforming growth factor (TGF-beta) was shown to be elevated in treated mice as early as 1 wk after the first instillation and increased gradually to 2.5 micrograms/lung at 3 wk (approximately 0.3 microgram/lung in saline-instilled controls). Intranasal instillation with F. rectivirgula was also associated with very significant increases in lung fibroblast collagen synthesis, starting at 2 wk. BAL macrophages from mice instilled with F. rectivirgula were found to release significantly more TGF-beta upon in vitro stimulation with zymosan beads than did BAL macrophages from saline-instilled mice. Finally, we show that supernatants from activated BAL macrophages of mice given F. rectivirgula increased quite significantly collagen synthesis in normal mouse lung fibroblasts. This increase could be abrogated by treating conditioned medium with a rabbit antibody against TGF-beta. Collectively, these data suggest that TGF-beta is generated in the course of experimental mouse hypersensitivity pneumonitis and contributes significantly to collagen synthesis.