l Title
Investigating the role of the epigenetic regulator G9a
in fibroblasts/epithelial cell crosstalk
l Problem and rationale for your research
Idiopathic pulmonary fibrosis (IPF) is characterized by an abnormal deposition of extracellular matrix proteins in the lung interstitium, leading to the accumulation of activated fibroblasts (myofibroblasts) and aberrant epithelial regeneration. Multiple signaling pathways have been implicated in IPF progression, yet epigenetic mechanisms underlying its persistent fibrogenic nature remain unknown. Epigenetic alterations, such as DNA methylation and histone modifications have been found to contribute to the development of numerous chronic diseases, including IPF (1-3). We have previously found that epigenetic mechanisms are directly responsible for perpetuating lung fibroblast activation by inhibiting the transcription of anti-fibrotic genes whose function is critical to maintaining fibroblasts in a quiescent state (4). Furthermore, we discovered that the epigenetic regulator homolog 5 (CBX5) and the histone methyltransferase EHMT (G9a) are critical to this process (4). Inhibition of CBX5/G9a pathway in IPF-derived lung fibroblast resulted in up-regulation of anti-fibrotic genes and fibroblast quiescence (4). Among the anti-fibrotic genes that are epigenetically repressed by G9a, we identified the mitochondrial regulator, PGC1α. Intriguingly, re-expression of PGC1a in IPF-derived lung fibroblasts led to their deactivation and quiescence, and this effect was accompanied by the inhibition of numerous secreted inflammatory and pro-fibrotic mediators, including IL-6, IL-8, CXCL5, and CXCL10. These findings suggest that abnormal epigenetic/metabolic circuits perpetuate pathogenic secretory activities in IPF fibroblasts resulting in paracrine cell activation and epithelial disrepair.
Recent studies have shown that aberrant epithelial-fibroblast interactions play an important role in the pathogenesis of IPF (5). However, the contribution of epigenetic-regulated fibroblast secretome to lung epithelial repair is currently unknown. In this study, we will focus on the effect of G9a-regulated secretome on fibroblast/epithelial cell communication.
l Details of the suggested approach
Here we will test the hypothesis that G9a overexpression in normal lung fibroblasts leads to the sustained release of inflammatory and pro-fibrotic mediators resulting in the disruption of lung epithelial regeneration and repair. To test this hypothesis, we have developed a tetracycline (Tet)-inducible G9a overexpression system (Fig.1A) to investigate fibroblast secretome and its contribution to lung epithelial cell expansion and differentiation. To this end, our laboratory has successfully developed ex vivo 3D organoid models to investigate epithelial expansion and differentiation, as well as fibroblast-epithelial communication.
First, to investigate the contribution of fibroblast G9a elevation to the secretion of inflammatory and profibrotic mediators, human lung fibroblasts infected with a control vector or those infected with a G9a vector will be treated with doxycycline for 48-72 hours to induce G9a overexpression. Our preliminary data shown in Fig.1B demonstrated robust elevation of G9a expression at 48 hours following doxycycline exposure. For this study, we chose 72 hours of incubation with doxycycline to allow the accumulation of secreted factors in the culture media. After 72 hours, we will evaluate the secretion of inflammatory and pro-fibrotic mediators by WB, ELISA, and cytokine array (R&D) (Fig.2A)
Next, to evaluate whether G9a elevation in lung fibroblasts affects epithelial expansion and differentiation, primary mouse lung epithelial cells will be isolated using magnetic beads (Miltenyibiotec) and co-cultured with control and G9-expressing human lung fibroblasts for 2 weeks to allow the formation of epithelial organoids (Fig.2B) During this time, co-cultured will be maintained in media containing doxycycline. The expansion of lung epithelial cells will be evaluated by measuring the colony-forming efficiency of the organoids. qPCR, immunohistochemistry, and immunofluorescence analysis will be performed at the end of the experiments to assess epithelial differentiation using specific markers, including SFTPC, PDBN (alveolar epithelial marker), SCGB1A, and FOXJ1 (airway marker). To confirm the fibroblast secretome, the conditioned medium will be collected from these organoids and tested using ELISA and secretory cytokine arrays, as previously described.
l How it will affect the broader field
It is well established that epithelial-fibroblast communication plays a role in lung regeneration following injury, and that this process is compromised during IPF progression. It is unknown, however, how epigenetically activated fibroblasts contribute to epithelial disrepair and disease progression. This study will contribute to our understanding of how the epigenetic regulator G9a influences fibroblasts and epithelial cell communication in IPF.
l References
1. Helling BA, Yang IV. Epigenetics in lung fibrosis: from pathobiology to treatment perspective. Curr Opin Pulm Med. 2015;21(5):454-62.
2. Barratt SL, Creamer A, Hayton C, Chaudhuri N. Idiopathic Pulmonary Fibrosis (IPF): An Overview. J Clin Med. 2018;7(8).
3. Glass DS, Grossfeld D, Renna HA, Agarwala P, Spiegler P, DeLeon J, et al. Idiopathic pulmonary fibrosis: Current and future treatment. Clin Respir J. 2022;16(2):84-96.
4. Ligresti G, Caporarello N, Meridew JA, Jones DL, Tan Q, Choi KM, et al. CBX5/G9a/H3K9me-mediated gene repression is essential to fibroblast activation during lung fibrosis. JCI Insight. 2019;5.
5. Hill C, Jones MG, Davies DE, Wang Y. Epithelial-mesenchymal transition contributes to pulmonary fibrosis via aberrant epithelial/fibroblastic cross-talk. J Lung Health Dis. 2019;3(2):31-5.