GRK2 promotes activation of lung fibroblast cells and contributes to pathogenesis of pulmonary fibrosis through increasing Smad3 expression

Pulmonary fibrosis is a chronic, progressive, and irreversible interstitial lung disease. Transforming growth factor beta1 (TGF-β1) plays a major role in lung fibroblast cell differentiation to myofibroblast cells and production of extracellular matrix, which are hallmarks of pulmonary fibrosis. G protein-coupled receptor kinase-2 (GRK2) has been shown to play controversial roles in TGF-β1-induced signal transduction in different cell types; however, the roles of GRK2 in TGF-β1-induced activation of lung fibroblast cells and development of pulmonary fibrosis have not been revealed. In this study, we found that GRK2 levels were induced in lungs and isolated fibroblast cells in a murine model of pulmonary fibrosis, as well as TGF-β1-treated lung fibroblasts. GRK2 levels were not changed in lungs in the injury phase of pulmonary fibrosis. Post-treatment with GRK2 inhibitor reduced ECM accumulation in lungs in bleomycin-challenged mice, suggesting that GRK2 activation contributes to the progressive phase of pulmonary fibrosis. Inhibition or downregulation of GRK2 attenuates fibronectin, collagen, and α-smooth muscle actin expression in TGF-β1-induced lung fibroblast cells or myofibroblast cells isolated from pulmonary fibrosis patients. Further, we showed that GRK2 regulates Smad3 expression, indicating that inhibition of GRK2 attenuates ECM accumulation through downregulation of Smad3 expression. This study reveals that GRK2 is a therapeutic target in treating pulmonary fibrosis and inhibition of GRK2 dampens pulmonary fibrosis by suppression of Smad3 expression, eventually attenuating TGF-β1 signal pathway and ECM accumulation.

HB-EGF-induced IL-8 secretion from airway epithelium leads to lung fibroblast proliferation and migration

Background We have reported that heparin-binding epidermal growth factor (HB-EGF) is increased in patients with chronic obstructive pulmonary disease (COPD) and associated with collagen deposition, but the mechanisms remain unclear. In the present study, we aimed to investigated the inflammatory cytokines secreted by bronchial epithelial cells following exposure to HB-EGF that promoted proliferation and migration of human lung fibroblast. Methods HB-EGF–induced inflammatory cytokines were assayed in two airway epithelial cells (primary human bronchial epithelial cells [HBECs] and BEAS-2B cells). Moreover, the culture supernatants derived from HB-EGF-treated HBECs and BEAS-2B cells were added to human primary lung fibroblasts. The effect of culture supernatants on proliferation and migration of fibroblasts was assessed. Results IL-8 expression was significantly increased in bronchial epithelial cells treated with HB-EGF, which was at least partially dependent on NF-kB pathways activation. HB-EGF–induced IL-8 was found to further promote lung fibroblasts proliferation and migration, and the effects were attenuated after neutralizing IL-8. Conclusions These findings suggest that HB-EGF may be involved in the pathology of airway fibrosis by induction of IL-8 from airway epithelium, subsequently causing lung fibroblasts proliferation and migration. Thus, inhibition of HBEGF and/or IL-8 production could prevent the development of airway fibrosis by modulating fibroblast activation.

Culture of IMR90 Cells in Advanced MEM with Reduced Serum

NOTE: Methods document and worksheet versions of this method are attached as PDFs.SCOPE OF APPLICATION (LIMITATIONS)This method describes the thawing, culturing, and cryopreservation of the human lung fibroblast cell line IMR90 in Advanced MEM-based growth medium. Disclaimer: The contents of this article have been reviewed by the US Environmental Protection Agency and approved for publication and do not necessarily represent Agency policy. Mention of trade names or commercial products does not constitute endorsement or recommendations for use.

Whole Transcriptome Analysis Revealed a Stress Response to Deep Underground Environment Conditions in Chinese Hamster V79 Lung Fibroblast Cells

Background: Prior studies have shown that the proliferation of V79 lung fibroblast cells could be inhibited by low background radiation (LBR) in deep underground laboratory (DUGL). In the current study, we revealed further molecular changes by performing whole transcriptome analysis on the expression profiles of long non-coding RNA (lncRNA), messenger RNA (mRNA), circular RNA (circRNA) and microRNA (miRNA) in V79 cells cultured for two days in a DUGL.Methods: Whole transcriptome analysis including lncRNA, mRNAs, circ RNA and miRNA was performed in V79 cells cultured for two days in DUGL and above ground laboratory (AGL), respectively. The differentially expressed (DE) lncRNA, mRNA, circRNA, and miRNA in V79 cells were identified by the comparison between DUGL and AGL groups. Quantitative real-time polymerase chain reaction(qRT-PCR)was conducted to verify the selected RNA sequencings. Then, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway was analyzed for the DE mRNAs which enabled to predict target genes of lncRNA and host genes of circRNA.Results: With |log2(Fold-change)| ≥ 1.0 and p < 0.05, a total of 1257 mRNAs (353 mRNAs up-regulated, 904 mRNAs down-regulated), 866 lncRNAs (145 lncRNAs up-regulated, 721 lncRNAs down-regulated), and 474 circRNAs (247 circRNAs up-regulated, 227 circRNAs down-regulated) were significantly altered between the two groups. There was no significant difference in miRNA between the two groups. The altered RNA profiles were mainly discovered in lncRNAs, mRNAs and circRNAs. DE RNAs were involved in many pathways including ECM-RI, PI3K-Akt signaling, RNA transport and the cell cycle under the LBR stress of the deep underground environment.Conclusion: Taken together, these results suggest that the LBR in the DUGL could induce transcriptional repression, thus reducing metabolic process and reprogramming the overall gene expression profile in V79 cells.

Extracellular HSP90α Interacts With ER Stress to Promote Fibroblasts Activation Through PI3K/AKT Pathway in Pulmonary Fibrosis

Pulmonary fibrosis is characterized by alveolar epithelial cell injury, lung fibroblast proliferation, differentiation, and extracellular matrix (ECM) deposition. Our previous study indicated that extracellular HSP90α (eHSP90α) promotes pulmonary fibrosis by activating the MAPK signaling pathway. Thus, treatment with 1G6-D7 (a selective HSP90α monoclonal antibody) to antagonize eHSP90α could effectively ameliorate fibrosis. This study aimed to elucidate the mechanism underlying the effects of eHSP90α in pulmonary fibrosis by focusing on its link with endoplasmic reticulum (ER) stress. Our results showed that eHSP90α promoted lung fibroblast differentiation by activating ER stress. Treatment with the ER stress inhibitor tauroursodeoxycholate (TUDCA) or glucose-regulated protein 78 kDa (GRP78) depletion significantly abrogated the effect of eHSP90α on ER stress and fibroblast activation. In addition, eHSP90α induced ER stress in fibroblasts via the phosphoinositide-4,5-bisphosphate 3-kinase (PI3K)-protein kinase B (AKT) signaling pathway, which could be blocked by the PI3K/AKT inhibitor LY294002, and blockade of eHSP90α by 1G6-D7 markedly inhibited ER stress in the model, indicating preventive and therapeutic applications. Intriguingly, we observed that TUDCA effectively reduced the secretion of eHSP90α in vitro and in vivo. In conclusion, this study shows that the interaction between eHSP90α and ER stress plays a crucial role in pulmonary fibrosis, indicating a positive feedback in lung fibroblasts. Targeting eHSP90α and alleviating fibroblast ER stress may be promising therapeutic approaches for pulmonary fibrosis.

DNA Methylation of Fibroblast Phenotypes and Contributions to Lung Fibrosis

Fibroblasts are an integral part of connective tissue and play a crucial role in developing and modulating the structural framework of tissues by acting as the primary source of extracellular matrix (ECM). A precise definition of the fibroblast remains elusive. Lung fibroblasts orchestrate the assembly and turnover of ECM to facilitate gas exchange alongside performing immune functions including the secretion of bioactive molecules and antigen presentation. DNA methylation is the covalent attachment of a methyl group to primarily cytosines within DNA. DNA methylation contributes to diverse cellular phenotypes from the same underlying genetic sequence, with DNA methylation profiles providing a memory of cellular origin. The lung fibroblast population is increasingly viewed as heterogeneous with between 6 and 11 mesenchymal populations identified across health and lung disease to date. DNA methylation has been associated with different lung fibroblast populations in health and with alterations in lung disease, but to varying extents. In this review, we will discuss lung fibroblast heterogeneity and the evidence for a contribution from DNA methylation to defining cell populations and alterations in disease.