LncRNA CTD-2528L19.6 prevents the progression of IPF by alleviating fibroblast activation

Long non-coding RNAs (lncRNAs) have emerged as critical factors for regulating multiple biological processes during organ fibrosis. However, the mechanism of lncRNAs in idiopathic pulmonary fibrosis (IPF) remains incompletely understood. In the present study, two sets of lncRNAs were defined: IPF pathogenic lncRNAs and IPF progression lncRNAs. IPF pathogenic and progression lncRNAs-mRNAs co-expression networks were constructed to identify essential lncRNAs. Network analysis revealed a key lncRNA CTD-2528L19.6, which was up-regulated in early-stage IPF compared to normal lung tissue, and subsequently down-regulated during advanced-stage IPF. CTD-2528L19.6 was indicated to regulate fibroblast activation in IPF progression by mediating the expression of fibrosis related genes LRRC8C, DDIT4, THBS1, S100A8 and TLR7 et al. Further studies showed that silencing of CTD-2528L19.6 increases the expression of Fn1 and Collagen I both at mRNA and protein levels, promoted the transition of fibroblasts into myofibroblasts and accelerated the migration and proliferation of MRC-5 cells. In contrast, CTD-2528L19.6 overexpression alleviated fibroblast activation in MRC-5 cells induced by TGF-β1. LncRNA CTD-2528L19.6 inhibited fibroblast activation through regulating the expression of LRRC8C in vitro assays. Our results suggest that CTD-2528L19.6 may prevent the progression of IPF from early-stage and alleviate fibroblast activation during the advanced-stage of IPF. Thus, exploring the regulatory effect of lncRNA CTD-2528L19.6 may provide new sights for the prevention and treatment of IPF.Subject terms: Mechanisms of disease, Non-coding RNAs     

Aortic Carboxypeptidase-like Protein (ACLP) Enhances Lung Myofibroblast Differentiation through Transforming Growth Factor β Receptor-dependent and -independent Pathways

Idiopathic pulmonary fibrosis (IPF) is a chronic and fatal lung disease characterized by the overgrowth, hardening, and scarring of lung tissue. The exact mechanisms of how IPF develops and progresses are unknown. IPF is characterized by extracellular matrix remodeling and accumulation of active TGFβ, which promotes collagen expression and the differentiation of smooth muscle α-actin (SMA)-positive myofibroblasts. Aortic carboxypeptidase-like protein (ACLP) is an extracellular matrix protein secreted by fibroblasts and myofibroblasts and is expressed in fibrotic human lung tissue and in mice with bleomycin-induced fibrosis. Importantly, ACLP knockout mice are significantly protected from bleomycin-induced fibrosis. The goal of this study was to identify the mechanisms of ACLP action on fibroblast differentiation. As primary lung fibroblasts differentiated into myofibroblasts, ACLP expression preceded SMA and collagen expression. Recombinant ACLP induced SMA and collagen expression in mouse and human lung fibroblasts. Knockdown of ACLP slowed the fibroblast-to-myofibroblast transition and partially reverted differentiated myofibroblasts by reducing SMA expression. We hypothesized that ACLP stimulates myofibroblast formation partly through activating TGFβ signaling. Treatment of fibroblasts with recombinant ACLP induced phosphorylation and nuclear translocation of Smad3. This phosphorylation and induction of SMA was dependent on TGFβ receptor binding and kinase activity. ACLP-induced collagen expression was independent of interaction with the TGFβ receptor. These findings indicate that ACLP stimulates the fibroblast-to-myofibroblast transition by promoting SMA expression via TGFβ signaling and promoting collagen expression through a TGFβ receptor-independent pathway.     

A Critical Role for the mTORC2 Pathway in Lung Fibrosis

A characteristic of dysregulated wound healing in IPF is fibroblastic-mediated damage to lung epithelial cells within fibroblastic foci. In these foci, TGF-β and other growth factors activate fibroblasts that secrete growth factors and matrix regulatory proteins, which activate a fibrotic cascade. Our studies and those of others have revealed that Akt is activated in IPF fibroblasts and it mediates the activation by TGF-β of pro-fibrotic pathways. Recent studies show that mTORC2, a component of the mTOR pathway, mediates the activation of Akt. In this study we set out to determine if blocking mTORC2 with MLN0128, an active site dual mTOR inhibitor, which blocks both mTORC1 and mTORC2, inhibits lung fibrosis. We examined the effect of MLN0128 on TGF-β-mediated induction of stromal proteins in IPF lung fibroblasts; also, we looked at its effect on TGF-β-mediated epithelial injury using a Transwell co-culture system. Additionally, we assessed MLN0128 in the murine bleomycin lung model. We found that TGF-β induces the Rictor component of mTORC2 in IPF lung fibroblasts, which led to Akt activation, and that MLN0128 exhibited potent anti-fibrotic activity in vitro and in vivo. Also, we observed that Rictor induction is Akt-mediated. MLN0128 displays multiple anti-fibrotic and lung epithelial-protective activities; it (1) inhibited the expression of pro-fibrotic matrix-regulatory proteins in TGF-β-stimulated IPF fibroblasts; (2) inhibited fibrosis in a murine bleomycin lung model; and (3) protected lung epithelial cells from injury caused by TGF-β-stimulated IPF fibroblasts. Our findings support a role for mTORC2 in the pathogenesis of lung fibrosis and for the potential of active site mTOR inhibitors in the treatment of IPF and other fibrotic lung diseases.     

Transforming Growth Factor-β1 Downregulates Vascular Endothelial Growth Factor-D Expression in Human Lung Fibroblasts via the Jun NH2-Terminal Kinase Signaling Pathway

Vascular endothelial growth factor (VEGF)-D, a member of the VEGF family, induces both angiogenesis and lymphangiogenesis by activating VEGF receptor-2 (VEGFR-2) and VEGFR-3 on the surface of endothelial cells. Transforming growth factor (TGF)-β1 has been shown to stimulate VEGF-A expression in human lung fibroblast via the Smad3 signaling pathway and to induce VEGF-C in human proximal tubular epithelial cells. However, the effects of TGF-β1 on VEGF-D regulation are unknown. To investigate the regulation of VEGF-D, human lung fibroblasts were studied under pro-fibrotic conditions in vitro and in idiopathic pulmonary fibrosis (IPF) lung tissue. We demonstrate that TGF-β1 downregulates VEGF-D expression in a dose- and time-dependent manner in human lung fibroblasts. This TGF-β1 effect can be abolished by inhibitors of TGF-β type I receptor kinase and Jun NH₂-terminal kinase (JNK), but not by Smad3 knockdown. In addition, VEGF-D knockdown in human lung fibroblasts induces G1/S transition and promotes cell proliferation. Importantly, VEGF-D protein expression is decreased in lung homogenates from IPF patients compared with control lung. In IPF lung sections, fibroblastic foci show very weak VEGF-D immunoreactivity, whereas VEGF-D is abundantly expressed within alveolar interstitial cells in control lung. Taken together, our data identify a novel mechanism for downstream signal transduction induced by TGF-β1 in lung fibroblasts, through which they may mediate tissue remodeling in IPF.     

Expression of WNT5A in Idiopathic Pulmonary Fibrosis and Its Control by TGF-β and WNT7B in Human Lung Fibroblasts

The wingless (Wnt) family of signaling ligands contributes significantly to lung development and is highly expressed in patients with usual interstitial pneumonia (UIP). We sought to define the cellular distribution of Wnt5A in the lung tissue of patients with idiopathic pulmonary fibrosis (IPF) and the signaling ligands that control its expression in human lung fibroblasts and IPF myofibroblasts. Tissue sections from 40 patients diagnosed with IPF or UIP were probed for the immunolocalization of Wnt5A. Further, isolated lung fibroblasts from normal or IPF human lungs, adenovirally transduced for the overexpression or silencing of Wnt7B or treated with TGF-β1 or its inhibitor, were analyzed for Wnt5A protein expression. Wnt5A was expressed in IPF lungs by airway and alveolar epithelium, smooth muscle cells, endothelium, and myofibroblasts of fibroblastic foci and throughout the interstitium. Forced overexpression of Wnt7B with or without TGF-β1 treatment significantly increased Wnt5A protein expression in normal human smooth muscle cells and fibroblasts but not in IPF myofibroblasts where Wnt5A was already highly expressed. The results demonstrate a wide distribution of Wnt5A expression in cells of the IPF lung and reveal that it is significantly increased by Wnt7B and TGF-β1, which, in combination, could represent key signaling pathways that modulate the pathogenesis of IPF.     

SPARC Suppresses Apoptosis of Idiopathic Pulmonary Fibrosis Fibroblasts through Constitutive Activation of ��-Catenin

Idiopathic pulmonary fibrosis (IPF) is a poorly understood progressive disease characterized by the accumulation of scar tissue in the lung interstitium. A hallmark of the disease is areas of injury to type II alveolar epithelial cells with attendant accumulation of fibroblasts in areas called fibroblastic foci. In an effort to better characterize the lung fibroblast phenotype in IPF patients, we isolated fibroblasts from patients with IPF and looked for activation of signaling proteins, which could help explain the exaggerated fibrogenic response in IPF. We found that IPF fibroblasts constitutively expressed increased basal levels of SPARC, plasminogen activator inhibitor-1 (PAI-1), and active β-catenin compared with control cells. Control of basal PAI-1 expression in IPF fibroblasts was regulated by SPARC-mediated activation of Akt, leading to inhibition of glycogen synthase kinase-3β and activation of β-catenin. Additionally, IPF fibroblasts (but not control fibroblasts) were resistant to plasminogen-induced apoptosis and were sensitized to plasminogen-mediated apoptosis by inhibition of SPARC or β-catenin. These findings uncover a newly discovered regulatory pathway in IPF fibroblasts that is characterized by elevated SPARC, giving rise to activated β-catenin, which regulates expression of downstream genes, such as PAI-1, and confers an apoptosis-resistant phenotype. Disruption of this pathway may represent a novel therapeutic target in IPF.     

Cartilage Oligomeric Matrix Protein in Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive and life threatening disease with median survival of 2.5–3 years. The IPF lung is characterized by abnormal lung remodeling, epithelial cell hyperplasia, myofibroblast foci formation, and extracellular matrix deposition. Analysis of gene expression microarray data revealed that cartilage oligomeric matrix protein (COMP), a non-collagenous extracellular matrix protein is among the most significantly up-regulated genes (Fold change 13, p-value <0.05) in IPF lungs. This finding was confirmed at the mRNA level by nCounter® expression analysis in additional 115 IPF lungs and 154 control lungs as well as at the protein level by western blot analysis. Immunohistochemical analysis revealed that COMP was expressed in dense fibrotic regions of IPF lungs and co-localized with vimentin and around pSMAD3 expressing cells. Stimulation of normal human lung fibroblasts with TGF-β1 induced an increase in COMP mRNA and protein expression. Silencing COMP in normal human lung fibroblasts significantly inhibited cell proliferation and negatively impacted the effects of TGF-β1 on COL1A1 and PAI1. COMP protein concentration measured by ELISA assay was significantly increased in serum of IPF patients compared to controls. Analysis of serum COMP concentrations in 23 patients who had prospective blood draws revealed that COMP levels increased in a time dependent fashion and correlated with declines in force vital capacity (FVC). Taken together, our results should encourage more research into the potential use of COMP as a biomarker for disease activity and TGF-β1 activity in patients with IPF. Hence, studies that explore modalities that affect COMP expression, alleviate extracellular matrix rigidity and lung restriction in IPF and interfere with the amplification of TGF-β1 signaling should be persuaded.     

NEDD4L-induced β-catenin ubiquitination suppresses the formation and progression of interstitial pulmonary fibrosis via inhibiting the CTHRC1/HIF-1α axis

Interstitial pulmonary fibrosis (IPF) is a severe progressive lung disease with limited therapeutic options and poor prognosis. Initially, we found the downregulated level of neural precursor cell expressed developmentally down-regulated 4-like protein (NEDD4L) in IPF-related expression microarray dataset, and this study was thus performed to explore the molecular mechanism of NEDD4L in IPF. The expression of NEDD4L was subsequently validated in lung tissues of IPF patients and mouse models. Then, mouse primary lung fibroblasts (LFs) were collected for in vitro functional experiments, with CCK-8, Transwell, and immunofluorescence assays used to examine the viability, migration, and differentiation of LFs. The in vitro findings were further assessed using in vivo mouse models. The expression of NEDD4L was down-regulated in lung tissues of IPF patients and mouse models. Overexpression of NEDD4L restricted the formation and progression of IPF in mice and attenuated the proliferative, invasive and differentiative abilities of LFs. Further, NEDD4L halted LFs activity by enhancing β-catenin ubiquitination and down-regulating the CTHRC1/HIF-1α axis. Also, in vivo experiments then validated that NEDD4L silencing repressed β-catenin ubiquitination and activated the CTHRC1/HIF-1α axis, thereby aggravating IPF in mice. NEDD4L may suppress the formation and progression of IPF through augmenting β-catenin ubiquitination and inhibiting the CTHRC1/HIF-1α axis.     

Fibroblast to myofibroblast transition is enhanced by increased cell density

Idiopathic pulmonary fibrosis (IPF) is a chronic disease of the lung caused by a rampant inflammatory response that results in the deposition of excessive extracellular matrix (ECM). IPF patient lungs also develop fibroblastic foci that consist of activated fibroblasts and myofibroblasts. In concert with ECM deposition, the increased cell density within fibroblastic foci imposes confining forces on lung fibroblasts. In this work, we observed that increased cell density increases the incidence of the fibroblast-to-myofibroblast transition (FMT), but mechanical confinement imposed by micropillars has no effect on FMT incidence. We found that human lung fibroblasts (HLFs) express more α-SMA and deposit more collagen matrix, which are both characteristics of myofibroblasts, in response to TGF-β1 when cells are seeded at a high density compared with a medium or a low density. These results support the hypothesis that HLFs undergo FMT more readily in response to TGF-β1 when cells are densely packed, and this effect could be dependent on increased OB-cadherin expression. This work demonstrates that cell density is an important factor to consider when modelling IPF in vitro, and it may suggest decreasing cell density within fibroblastic foci as a strategy to reduce IPF burden.     

Calcium and integrin binding protein 1 (CIB1) induces myocardial fibrosis in myocardial infarction via regulating the PI3K/Akt pathway

Myocardial infarction (MI) is a severe coronary artery disease resulted from substantial and sustained ischemia. Abnormal upregulation of calcium and integrin binding protein 1 (CIB1) has been found in several cardiovascular diseases. In this study, we established a mouse model of MI by permanent ligation of the left anterior descending coronary artery. CIB1 was upregulated in the heart of MI mice. Notably, CIB1 knockdown by intramuscular injection of lentivirus-mediated short hairpin RNA (shRNA) targeting Cib1 improved cardiac function and attenuated myocardial hypertrophy and infarct area in MI mice. MI-induced upregulation of α-SMA, vimentin, Collagen I, and Collagen III, which resulted in collagen production and myocardial fibrosis, were regressed by CIB1 silencing. In vitro, cardiac fibroblasts (CFs) isolated from mice were subjected to angiotensin II (Ang II) treatment. Inhibition of CIB1 downregulated the expression of α-SMA, vimentin, Collagen I, and Collagen III in Ang II-treated CFs. Moreover, CIB1 knockdown inhibited Ang II-induced phosphorylation of PI3K-p85 and Akt in CFs. The effect of CIB1 knockdown on Ang II-induced cellular injury was comparable to that of LY294002, a specific inhibitor of the PI3K/Akt pathway. We demonstrated that MI-induced cardiac hypertrophy, myocardial fibrosis, and cardiac dysfunction might be attributed to the upregulation of CIB1 in MI mice. Downregulation of CIB1 alleviated myocardial fibrosis and cardiac dysfunction by decreasing the expression of α-SMA, vimentin, Collagen I, and Collagen III via inhibiting the PI3K/Akt pathway. Therefore, CIB1 may be a potential target for MI treatment.     

Suppression of lncRNA NLRP3 inhibits NLRP3-triggered inflammatory responses in early acute lung injury

Acute lung injury (ALI) is a common lung pathology that is accompanied by alveolar macrophage (AM) activation and inflammatory response. This study investigated the role of the long non-coding RNA NONRATT004344 (hereafter named lncRNA NLRP3) in regulating the Nod-like receptor protein 3 (NLRP3)-triggered inflammatory response in early ALI and the underlying mechanism as well. We established LPS-induced ALI models to explore their interactive mechanisms in vitro and in vivo. Luciferase reporter assays were performed to determine that miR-138-5p could bind to lncRNA NLRP3 and NLRP3. We observed increased lncRNA NLRP3 expression, decreased miR-138-5p expression, NLRP3 inflammasome activation, and upregulated caspase-1, IL-1β, and IL-18 expression in the LPS-induced ALI model. Furthermore, lncRNA NLRP3 overexpression activated the NLRP3 inflammasome and promoted IL-1β and IL-18 secretion; the miR-138-5p mimic abolished these effects in vivo and in vitro. Consistently, miR-138-5p inhibition reversed the effects of lncRNA NLRP3 silencing on the expression of NLRP3-related molecules and inhibition of the NLRP3/caspase-1/IL-1β signalling pathway. Mechanistically, lncRNA NLRP3 sponging miR-138-5p facilitated NLRP3 activation through a competitive endogenous RNA (ceRNA) mechanism. In summary, our results suggested that lncRNA NLRP3 binding miR-138-5p promotes NLRP3-triggered inflammatory response via lncRNA NLRP3/miR-138-5p/NLRP3 ceRNA network (ceRNET) and provides insights into the treatment of early ALI.Subject terms: Bacterial infection, Inflammasome     

Plasminogen Activator Inhibitor-1 Suppresses Profibrotic Responses in Fibroblasts from Fibrotic Lungs

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by progressive interstitial scarification. A hallmark morphological lesion is the accumulation of myofibroblasts or fibrotic lung fibroblasts (FL-fibroblasts) in areas called fibroblastic foci. We previously demonstrated that the expression of both urokinase-type plasminogen activator (uPA) and the uPA receptor are elevated in FL-fibroblasts from the lungs of patients with IPF. FL-fibroblasts isolated from human IPF lungs and from mice with bleomycin-induced pulmonary fibrosis showed an increased rate of proliferation compared with normal lung fibroblasts (NL-fibroblasts) derived from histologically “normal” lung. Basal expression of plasminogen activator inhibitor-1 (PAI-1) in human and murine FL-fibroblasts was reduced, whereas collagen-I and α-smooth muscle actin were markedly elevated. Conversely, alveolar type II epithelial cells surrounding the fibrotic foci in situ, as well as those isolated from IPF lungs, showed increased activation of caspase-3 and PAI-1 with a parallel reduction in uPA expression. Transduction of an adenovirus PAI-1 cDNA construct (Ad-PAI-1) suppressed expression of uPA and collagen-I and attenuated proliferation in FL-fibroblasts. On the contrary, inhibition of basal PAI-1 in NL-fibroblasts increased collagen-I and α-smooth muscle actin. Fibroblasts isolated from PAI-1-deficient mice without lung injury also showed increased collagen-I and uPA. These changes were associated with increased Akt/phosphatase and tensin homolog proliferation/survival signals in FL-fibroblasts, which were reversed by transduction with Ad-PAI-1. This study defines a new role of PAI-1 in the control of fibroblast activation and expansion and its role in the pathogenesis of fibrosing lung disease and, in particular, IPF.     

Transglutaminase 2: a novel therapeutic target for idiopathic pulmonary fibrosis using selective small molecule inhibitors

This study investigates the effects of a site-directed TG2-selective inhibitor on the lung myofibroblast phenotype and ECM deposition to elucidate TG2 as a novel therapeutic target in idiopathic pulmonary fibrosis (IPF)—an incurable progressive fibrotic disease. IPF fibroblasts showed increased expression of TG2, α smooth muscle actin (αSMA) and fibronectin (FN) with increased extracellular TG2 and transforming growth factor β1 (TGFβ1) compared to normal human lung fibroblasts (NHLFs) which do not express αSMA and express lower levels of FN. The myofibroblast phenotype shown by IPF fibroblasts could be reversed by selective TG2 inhibition with a reduction in matrix FN and TGFβ1 deposition. TG2 transduction or TGFβ1 treatment of NHLFs led to a comparable phenotype to that of IPF fibroblasts which was reversible following selective TG2 inhibition. Addition of exogenous TG2 to NHLFs also induced the myofibroblast phenotype by a mechanism involving TGFβ1 activation which could be ameliorated by selective TG2 inhibition. SMAD3-deleted IPF fibroblasts via CRISPR-cas9 genome editing, showed reduced TG2 protein levels following TGFβ1 stimulation. This study demonstrates a key role for TG2 in the induction of the myofibroblast phenotype and shows the potential for TG2-selective inhibitors as therapeutic agents for the treatment of fibrotic lung diseases like IPF.

     

Novel lncRNA lncRNA001074 participates in the low salinity–induced response in the sea cucumber Apostichopus japonicus by targeting the let-7/NKAα axis

Salinity fluctuations have severe impacts on sea cucumbers and therefore important consequences in sea cucumber farming. The responses of sea cucumbers to salinity changes are reflected in the expression profiles of multiple genes and non-coding RNAs (ncRNAs). The microRNA (let-7) which is a developmental regulator, the ion transporter gene sodium potassium ATPase gene (NKAα), and the long ncRNA lncRNA001074 were previously shown to be involved in responses to salinity changes in various marine species. To better understand the relationship between ncRNAs and target genes, the let-7/NKAα/lncRNA001074 predicted interaction was investigated in this study using luciferase reporter assays and gene knockdowns in the sea cucumber Apostichopus japonicus. The results showed that NKAα was the target gene of let-7 and NKAα expression levels were inversely correlated with let-7 expression based on the luciferase reporter assays and western blots. The let-7 abundance was negatively regulated by lncRNA001074 and NKAα both in vitro and in vivo. Knockdown of lncRNA001074 led to let-7 overexpression. These results demonstrated that lncRNA001074 binds to the 3′-UTR binding site of let-7 in a regulatory manner. Furthermore, the expression profiles of let-7, NKAα, and lncRNA001074 were analyzed in sea cucumbers after the knockdown of each of these genes. The results found that lncRNA001074 competitively bound let-7 to suppress NKAα expression under low salinity conditions. The downregulation of let-7, in conjunction with the upregulation of lncRNA001074 and NKAα, may be essential for the response to low salinity change in sea cucumbers. Therefore, the dynamic balance of the lncRNA001074, NKAα, and let-7 network might be a potential response mechanism to salinity change in sea cucumbers.