RATIONALE: Mesenchymal progenitor cells (MPCs) in the lungs of individuals with Idiopathic Pulmonary Fibrosis (IPF) show intrinsic fibrogenic properties, possessing a distinct transcriptomic profile and maintaining fibrogenic activity, which contributes to the fibroblast population in IPF. We hypothesize that IPF MPCs are distinguished from control MPCs by a unique gene interaction network. METHODS: We analyzed bulk RNA-seq data, available on the Gene Expression Omnibus (GEO) under accession numbers GSE97038 and GSE276568. In these experiments, CD44hi MPCs were isolated through flow cytometry from mesenchymal cell lines derived from IPF patients (explanted lungs from live IPF patients after lung transplant) and control subjects (lobectomy for clinical reasons) using antibodies against CD44 and SSEA4. The pooled dataset comprises nine IPF and nine control MPC lines. We identified differentially expressed genes (DEGs) in IPF MPCs compared to controls using DESeq and performed pathway enrichment analysis on DEGs using Gene Ontology (GO). We also conducted Gene Set Enrichment Analysis (GSEA) with gene sets related to stemness, pluripotency, and self-renewal. Weighted gene co-expression network analysis (WGCNA) identified functional modules specifically associated with IPF MPCs. RESULTS: Applying an adjusted p-value threshold of 0.05, we identified 134 DEGs in IPF MPCs compared to controls. GO analysis showed significant enrichment in pathways associated with leukocyte migration, extracellular matrix organization, and cell differentiation and growth. GSEA highlighted significant enrichment in two gene sets related to stemness, with adjusted p-values of 0.002 and 0.048. Using WGCNA, we identified 16 functional modules, one of which reached significance based on an adjusted p-value threshold of 0.05 and a correlation coefficient cutoff of 0.45. This significant module demonstrated a positive correlation with IPF status (correlation coefficient of 0.69, adjusted p-value 0.0085). CONCLUSION: This study is the first to characterize a distinct gene expression profile in IPF MPCs compared to controls, highlighting enriched biological processes relevant to IPF pathogenesis. GSEA results further support the hypothesis that IPF MPCs display stem cell-like gene expression patterns, including genes associated with pluripotency and self-renewal. Additionally, we identified functional gene modules that significantly correlate with the fibrogenic properties of IPF MPCs, including hub genes that may play critical roles in promoting fibrosis in IPF. These findings deepen our understanding of IPF MPC biology and identify potential targets for future research. We plan to expand this approach by increasing the sample size to enhance statistical power, aiming to identify more robust functional modules and their highly connected hub genes.

Our research offers valuable understanding regarding the epigenetic control of IPF MPC. The data we obtained strongly support the idea that the coordination between chromatin remodeling and histone methylation plays a key role in regulating transcription factors. Specifically, our findings indicate that FOXO1, an essential transcription factor, likely governs the self-renewal of IPF MPC, which is crucial for maintaining a critical pool of fibrogenic MPCs. This interplay could be an important therapeutic target.

The idiopathic pulmonary fibrosis (IPF) lung contains mesenchymal progenitor cells (MPCs) that display durable activation of oncogenic signaling and cell-autonomous fibrogenicity in vivo. Prior work identified a CD44/Brg1/PRMT5 nuclear regulatory module in IPF MPCs that increased the expression of genes positively regulating pluripotency and self-renewal. Left unanswered is how IPF MPCs evade negative regulation of self-renewal. Here we sought to identify mechanisms disabling negative regulation of self-renewal in IPF MPCs. We demonstrate that expression of the tumor suppressor genes rbl1 and pten is decreased in IPF MPCs. The mechanism involves the CD44-facilitated association of the chromatin remodeler Brg1 with the histone-modifying methyltransferase PRMT5. Brg1 enhances chromatin accessibility leading to PRMT5-mediated methylation of H3R8 and H4R3 on the rbl1 and pten genes, repressing their expression. Genetic knockdown or pharmacological inhibition of either Brg1 or PRMT5 restored RBL1 and PTEN expression reduced IPF MPC self-renewal in vitro and inhibited IPF MPC-mediated pulmonary fibrosis in vivo. Our studies indicate that the CD44/Brg1/PRMT5 regulatory module not only functions to activate positive regulators of pluripotency and self-renewal but also functions to repress tumor suppressor genes rbl1 and pten. This confers IPF MPCs with the cancer-like property of cell-autonomous self-renewal providing a molecular mechanism for relentless fibrosis progression in IPF. NEW & NOTEWORTHY Here we demonstrate that a CD44/Brg1/PRMT5 epigenetic regulatory module represses the tumor suppressor genes RBL1 and PTEN in IPF mesenchymal progenitor cells, thereby promoting their self-renewal and maintenance of a critical pool of fibrogenic mesenchymal progenitor cells.

Supplementary Table 5 from Eukaryotic Translation Initiation Factor 4E–Induced Progression of Primary Human Mammary Epithelial Cells along the Cancer Pathway Is Associated with Targeted Translational Deregulation of Oncogenic Drivers and Inhibitors

Supplementary Figure 2 from Eukaryotic Translation Initiation Factor 4E–Induced Progression of Primary Human Mammary Epithelial Cells along the Cancer Pathway Is Associated with Targeted Translational Deregulation of Oncogenic Drivers and Inhibitors

Supplementary Fig. S4. Over expressed eIF4E induces luminal subtype tumors.

Supplementary Fig. S2. HA-eIF4E-induced pre-malignant lesions persist in post-weaning mammary glands.

Here we show that IL-8 concurrently promotes senescence and upregulation of PD-L1 in IPF MPCs. IHC analysis identifies the presence of senescent IPF MPCs intermingled with NK cells in the fibroblastic focus, suggesting that senescent MPCs elude immune cell surveillance. We demonstrate that disruption of PD-1/PD-L1 interaction promotes NK cell killing of IPF MPCs and arrests IPF MPC-mediated experimental lung fibrosis. Disruption of PD-1/PD-L1 interaction may be one means to limit fibrotic progression.

Supplementary Fig. S3. Double K8/Ki-67staining of wild type (WT) and transgenic (WAP-4E) mammary glands (gestation 6).

Supplementary Figure 3 from Eukaryotic Translation Initiation Factor 4E–Induced Progression of Primary Human Mammary Epithelial Cells along the Cancer Pathway Is Associated with Targeted Translational Deregulation of Oncogenic Drivers and Inhibitors

PDF file - 32K, Recognition of capped mRNA for translation initiation by the complex eIF4F. The eukaryotic translation initiation factor 4E (eIF4E), a 25 kDa phosphoprotein which directly contact to the cap structure, exists either as a monomer or as a subunit of the translation initiation complex eIF4F. When bound to capped messages, eIF4E control post-transcriptional gene expression in the nucleus, where it facilitates the nuclear export of some mRNAs, and in the cytoplasm, where it selectively activates mRNAs for their recruitment to ribosomes. The trimolecular complex eIF4F is essential for recruitment of capped transcripts to ribosomes and subsequent ribosome scanning. It consists of translational factors eIF4E, eIF4G and eIF4A and for the eIF4A cofactor eIF4B. There are two isoforms of translation initiation factor 4G, eIF4GI and eIF4GII that serve a docking function, with the amino terminal half binding to eIF4E, and the C-terminal half binding to eIF4A. The eIF4G family proteins also have recognition sites for several other peptides including for the poly (A) binding protein (PARP), which facilitates circularization of the eIF4F-mRNA complex. Translation initiation factor 4A functions as an ATP requiring helicase by unwinding the 5' region

Hypoxia is a sentinel feature of idiopathic pulmonary fibrosis (IPF). The IPF microenvironment contains high lactate levels, and hypoxia enhances cellular lactate production. Lactate, acting through the GPR81 lactate receptor, serves as a signal molecule regulating cellular processes. We previously identified intrinsically fibrogenic mesenchymal progenitor cells (MPCs) that drive fibrosis in the lungs of patients with IPF. However, whether hypoxia enhances IPF MPC fibrogenicity is unclear. We hypothesized that hypoxia increases IPF MPC fibrogenicity via lactate and its cognate receptor GPR81. Here we show that hypoxia promotes IPF MPC self-renewal. The mechanism involves hypoxia-mediated enhancement of LDHA function and lactate production and release. Hypoxia also increases HIF1α levels, and this increase in turn augments the expression of GPR81. Exogenous lactate operating through GPR81 promotes IPF MPC self-renewal. IHC analysis of IPF lung tissue demonstrates IPF MPCs expressing GPR81 and hypoxic markers on the periphery of the fibroblastic focus. We show that hypoxia enhances IPF MPC fibrogenicity in vivo. We demonstrate that knockdown of GPR81 inhibits hypoxia-induced IPF MPC self-renewal in vitro and attenuates hypoxia-induced IPF MPC fibrogenicity in vivo. Our data demonstrate that hypoxia creates a feed-forward loop that augments IPF MPC fibrogenicity via the lactate/GPR81/HIF1α pathway.

Supplementary Fig. S1. Generation of WAP-4E transgenic mice.

Supplementary Table 6 from Eukaryotic Translation Initiation Factor 4E–Induced Progression of Primary Human Mammary Epithelial Cells along the Cancer Pathway Is Associated with Targeted Translational Deregulation of Oncogenic Drivers and Inhibitors

Supplementary Table 4 from Eukaryotic Translation Initiation Factor 4E–Induced Progression of Primary Human Mammary Epithelial Cells along the Cancer Pathway Is Associated with Targeted Translational Deregulation of Oncogenic Drivers and Inhibitors

Supplementary Figure 1 from Eukaryotic Translation Initiation Factor 4E–Induced Progression of Primary Human Mammary Epithelial Cells along the Cancer Pathway Is Associated with Targeted Translational Deregulation of Oncogenic Drivers and Inhibitors

PDF file - 59K, Recognition of the cap

Supplementary Fig. S5. HA-eIF4E rescues HMEC/hTERT cells from Ras-induced senescence and activates ATR signaling.

Supplementary Table 9 from Eukaryotic Translation Initiation Factor 4E–Induced Progression of Primary Human Mammary Epithelial Cells along the Cancer Pathway Is Associated with Targeted Translational Deregulation of Oncogenic Drivers and Inhibitors

Supplementary Table 2 from Eukaryotic Translation Initiation Factor 4E–Induced Progression of Primary Human Mammary Epithelial Cells along the Cancer Pathway Is Associated with Targeted Translational Deregulation of Oncogenic Drivers and Inhibitors