Distinct roles of KLF4 in mesenchymal cell subtypes during lung fibrogenesis (original) (raw)
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2022
There is a paucity of information about potential molecular brakes on the activation of fibroblasts that drive tissue fibrosis. The transcription factor Kruppel-like factor 4 (KLF4) is best known as a determinant of cell stemness and a tumor suppressor. We found that its expression was diminished in fibroblasts from fibrotic lung. Gain- and loss-of-function studies showed that KLF4 inhibits fibroblast proliferation, collagen synthesis, and differentiation to myofibroblasts, while restoring their sensitivity to apoptosis. Conditional deletion of KLF4 from fibroblasts potentiated the peak degree of pulmonary fibrosis and abrogated the subsequent spontaneous resolution that follows in a model of transient fibrosis. A small molecule inducer of KLF4 was able to restore its expression in fibrotic fibroblasts and elicit resolution in an experimental model characterized by more clinically relevant persistent pulmonary fibrosis. These data identify KLF4 as a pivotal brake on fibroblast activ...
Molecular biology of the cell, 2017
The contribution of epithelial-to-mesenchymal transition (EMT) to the pro-fibrotic stiff microenvironment and myofibroblast accumulation in pulmonary fibrosis remains unclear. We examined EMT-competent lung epithelial cells and lung fibroblasts from control (fibrosis-free) donors or patients with idiopathic pulmonary fibrosis (IPF), which is a very aggressive fibrotic disorder. Cells were cultured on pro-fibrotic conditions including stiff substrata and TGF-β1, and analyzed in terms of morphology, stiffness and expression of EMT/myofibroblast markers and fibrillar collagens. All fibroblasts acquired a robust myofibroblast phenotype upon TGF-β1 stimulation. Yet, IPF-myofibroblasts exhibited higher stiffness and expression of fibrillar collagens than control fibroblasts, concomitantly with enhanced FAK(Y397) activity. FAK inhibition was sufficient to decrease fibroblast stiffness and collagen expression, supporting that FAK(Y397) hyperactivation may underlie the aberrant mechanobiolog...
ELECTROPHORESIS, 2003
Cell cultures of mesenchymal type were obtained from biopsies taken after bronchoscopy from patients with asthma. It was possible to achieve outgrowth of fibroblastlike cells from these lung biopsies, which stained for a-smooth actin indicating that they were of myofibroblast type. Morphologically, two types of myofibroblasts could be observed: one intermediate form with more stretched cell shape and lamellipodia protrusions, and one more differentiated compact form of myofibroblast. The intermediate form was the most dominant type in these patients, indicating an active ongoing remodelling process. Further studies showed that platelet-derived growth factor (PDGF) might be the factor that stimulates the formation of the intermediate type of myofibroblasts, since it enhance migration of normal human lung fibroblasts 4-fold compared to control through an induced formation of stress fibers and lamellipodia protrusions. Additionally, intracellular signalling pathways involved in migration, such as RhoA and MAPkinase were stimulated 1.5-fold and 3.5-fold, respectively. By using two-dimensional (2-D) gel electrophoresis and protein identification by peptide mass finger printing matrix assisted laser desporption/ionization -time of flight -massspectrometry (MALDI-TOF-MS) it was possible to confirm that PDGF affected the synthesis of proteins involved in the remodelling process, such as collagen VI and posttranslational forms thereof. PDGF also stimulated the production of FK506 binding protein of 65 kDa, a protein involved in smooth muscle differentiaition, and proteins involved in the rearrangement of the cytoskeleton connected to migration such as the actin related protein ARP3, the T-complex protein and the heat shock protein 60. We demonstrate that PDGF has a potential pathological role in asthma and formation of subepithelial fibrosis by inducing changes in the proteome.
Journal of Biological Chemistry, 2013
Background: Dysregulated fibroblast-to-myofibroblast transitions cause fibrotic diseases. Results: Aortic carboxypeptidase-like protein (ACLP) stimulates myofibroblast differentiation through activation of transforming growth factor  receptor signaling. Conclusion: Eliminating ACLP activity reduces SMA expression and myofibroblast formation. Significance: Reducing ACLP function in fibrotic tissue may provide a novel strategy to reduce the rate of fibrotic disease progression. 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 TGF, which promotes collagen expression and the differentiation of smooth muscle ␣-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 TGF signaling. Treatment of fibroblasts with recombinant ACLP induced phosphorylation and nuclear translocation of Smad3. This phosphorylation and induction of SMA was dependent on TGF receptor binding and kinase activity. ACLP-induced collagen expression was independent of interaction with the TGF receptor. These findings indicate that ACLP stimulates the fibroblast-to-myofibroblast transition by promoting SMA expression via TGF signaling and promoting collagen expression through a TGF receptor-independent pathway. Idiopathic pulmonary fibrosis (IPF) 3 is a chronic lung disease with no effective cure (1). It is characterized by sequential lung injury that results in epithelial and endothelial cell damage, inflammation, and progressive deposition of ECM molecules, including collagen. Fibrotic lungs contain large numbers of contractile, filament-laden parenchymal cells known as myofibroblasts and an increase in overall tissue contractility (2). These myofibroblasts characteristically express smooth muscle ␣-actin (SMA) and are known to be critical components of wound healing (2, 3). Myofibroblasts originate from different sources, including fibroblasts, epithelial cells, and bone marrow-derived cells (4-8). Myofibroblasts are spindle-or stellate-shaped cells that are similar to smooth muscle cells in that they are both contractile and contain SMA (2). Differentiated myofibroblasts are also responsible for increased collagen synthesis in the lung in IPF (2). In the fibrotic lung, normal fibroblastic cells become activated by cytokines released from local inflammatory and resident cells after tissue injury to promote ECM component synthesis (9, 10). Mechanical challenges in the extracellular environment also stimulate the fibroblast-to-myofibroblast transition. In response to mechanical challenges, these cells develop stress fibers that connect the cell to ECM proteins (11). Along with extracellular remodeling, accumulation of active TGF and the presence of ECM proteins like the extra domain A (ED-A) splice variant of fibronectin are required events for the production of SMA-positive myofibroblasts (9). TGF is found at high levels in human IPF lungs (12) as well as in the lungs of mice and hamsters with bleomycin-induced fibrosis (13, 14). TGF overexpression is sufficient to induce fibrosis in rat lungs (15). Additionally, TGF promotes the formation of granulation tissue with abundant SMA-expressing myofibroblasts in rats and induces SMA expression in cultured fibroblasts (16). TGF has also been shown to activate the transcriptional regulator myocardin-related transcription factor A * This work was supported, in whole or in part, by National Institutes of Health Grants HL078869 and HL078869-04S1 (to M. D. L.). This work was also supported by Department of Biochemistry start-up funds.
Journal of Translational Medicine
Background: Neurotrophins (NT) belongs to a family of growth factors which promotes neurons survival and differentiation. Increasing evidence show that NT and their receptor are expressed in lung tissues suggesting a possible role in lung health and disease. Here we investigated the expression and functional role of the TrkB/BDNF axis in idiopathic pulmonary fibrotic lung (myo)fibroblasts. Methods: Lung fibroblast were isolated from IPF patients and characterized for the expression of mesenchymal markers in comparison to normal lung fibroblasts isolated from non-IPF controls. Results: BDNF treatment promoted mesenchymal differentiation and this effect was counteracted by the TrkB inhibitor K252a. In this regard, we showed that K252a treatment was able to control the expression of transcription factors involved in epithelial to mesenchymal transition (EMT). Accordingly, K252a treatment reduced matrix metalloproteinase-9 enzyme activity and E-cadherin expression while increased cytoplasmic β-catenin expression. Conclusions: Our results suggest that BDNF/TrkB axis plays a role in EMT promoting the acquisition of (myo) fibroblast cell phenotype in IPF. Targeting BDNF/TrkB seems to represent a viable approach in order to prevent EMT dependent lung fibrosis.
Lack of MK2 Inhibits Myofibroblast Formation and Exacerbates Pulmonary Fibrosis
2007
Fibroblasts play a major role in tissue repair and remodeling. Their differentiation into myofibroblasts, marked by increased expression of smooth muscle-specific a-actin (a-SMA), is believed to be important in wound healing and fibrosis. We have recently described a role for MK2 in this phenotypic differentiation in culture. In this article, we demonstrate that MK2 also regulates myofibroblasts in vivo. Disruption of MK2 in mice prevented myofibroblast formation in a model of pulmonary fibrosis. However, MK2 disruption and consequent lack of myofibroblast formation exacerbated fibrosis rather than ameliorated it as previously postulated. When mice lacking MK2 (MK2 2/2 ) were exposed to bleomycin, more collagen accumulated and more fibroblasts populated fibrotic regions in their lungs than in similarly treated wild-type mice. While there were many vimentin-positive cells in the bleomycin-treated MK2 2/2 mouse lungs, few a-SMA-positive cells were observed in these lungs compared with wild-type mouse lungs. siRNA against MK2 reduced a-SMA expression in wild-type mouse embryonic fibroblasts (MEF), consistent with its suppression in MK2 2/2 MEF. On the other hand expressing constitutively active MK2 in MK2 2/2 MEF significantly increased a-SMA expression. MK2 2/2 MEF proliferated at a faster rate and produced more collagen; however, they migrated at a slower rate than wild-type MEF. Overexpressing phosphomimicking HSP27, a target of MK2, did not reverse the effect of MK2 disruption on fibroblast migration. MK2 disruption did not affect Smad2 activation by transforming growth factor-b. Thus, MK2 appears to mediate myofibroblast differentiation, and inhibiting that differentiation might contribute to fibrosis rather than protect against it.
FGF-1 Activates several Signaling Pathways as an Antifibrogenic Mediator in Human Lung Fibroblasts
2020
Background: In idiopathic pulmonary fibrosis (IPF), fibroblasts characteristically perform abnormal turnover of the extracellular matrix by downregulating matrix metalloproteinase-1 (MMP-1) and upregulating collagen I and alpha-smooth muscle actin (α-SMA), mostly through the stimulation of transforming growth factor beta-1 (TGF-β1). Alternatively, fibroblast growth factor-1 combined with heparin (FGF-1/H) inhibits these effects. In view of this, the aim of this research was to determine which signaling pathways stimulated by FGF1/H in fibroblasts upregulate MMP-1 and downregulate collagen I and TGF-β1-induced α-SMA expression in human lung fibroblasts (HLF). Methods: Cells were stimulated with FGF-1/H in the presence or absence of selective pharmacological inhibitors of different signaling pathways to block MMP-1, collagen I, and TGF-β1-induced α-SMA expression. Expression levels of the aforementioned proteins and genes were evaluated by RT-qPCR and Western blot. Results: Data showe...