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Project description:RNA-binding proteins control gene expression in cardiac fibroblasts during TGFb-driven fibrotic responses, including the eukaryotic translation initiation factor 4G2 (eIF4G2), which was known to facilitate alternative mRNA translation during cellular stresses. However, the precise role of eIF4G2 in cardiac fibroblast activation in response to fibrotic stress remains unclear. In this study, we found increased eIF4G2 protein levels in the hearts of heart failure patients and myocardial infarcted mice, as well as in TGFb-treated immortalized human and primary mouse cardiac fibroblasts. Depletion of eIF4G2 led to predominant translational downregulation of genes that are enriched in focal adhesion and extracellular matrix pathways. eIF4G2 knockdown reduced the proliferation, migration, and collagen secretion of TGFb-treated cardiac fibroblasts. Mechanistically, we found that the translation of IGFBP7 mRNA relies on eIF4G2, which is crucial for ECM production in response to pro-fibrotic stimuli. The interaction between eIF4G2 and a DEAD box RNA helicase DDX24 can regulate IGFBP7 protein expression in cardiac fibroblasts. Together, these findings suggest that the TGFb-eIF4G2-IGFBP7 axis establishes a novel translational regulatory circuit that governs cardiac fibroblast activation. Furthermore, conditional knockout of Eif4g2 in POSTN-positive myofibroblasts reduced cardiac fibrosis in the myocardial infarction mouse model, thus improving cardiac function. Our study provides insights into the molecular mechanism of eIF4G2-mediated translational regulation of crucial physiological mRNAs during cardiac fibroblast activation.
2024-07-31 | GSE239824 | GEO
Project description:RNA-binding proteins control gene expression in cardiac fibroblasts during TGFb-driven fibrotic responses, including the eukaryotic translation initiation factor 4G2 (eIF4G2), which was known to facilitate alternative mRNA translation during cellular stresses. However, the precise role of eIF4G2 in cardiac fibroblast activation in response to fibrotic stress remains unclear. In this study, we found increased eIF4G2 protein levels in the hearts of heart failure patients and myocardial infarcted mice, as well as in TGFb-treated immortalized human and primary mouse cardiac fibroblasts. Depletion of eIF4G2 led to predominant translational downregulation of genes that are enriched in focal adhesion and extracellular matrix pathways. eIF4G2 knockdown reduced the proliferation, migration, and collagen secretion of TGFb-treated cardiac fibroblasts. Mechanistically, we found that the translation of IGFBP7 mRNA relies on eIF4G2, which is crucial for ECM production in response to pro-fibrotic stimuli. The interaction between eIF4G2 and a DEAD box RNA helicase DDX24 can regulate IGFBP7 protein expression in cardiac fibroblasts. Together, these findings suggest that the TGFb-eIF4G2-IGFBP7 axis establishes a novel translational regulatory circuit that governs cardiac fibroblast activation. Furthermore, conditional knockout of Eif4g2 in POSTN-positive myofibroblasts reduced cardiac fibrosis in the myocardial infarction mouse model, thus improving cardiac function. Our study provides insights into the molecular mechanism of eIF4G2-mediated translational regulation of crucial physiological mRNAs during cardiac fibroblast activation.
2024-07-31 | GSE239823 | GEO
Project description:BACKGROUND:Fibroblast apoptosis is a critical component of normal repair and the acquisition of an apoptosis-resistant phenotype contributes to the pathogenesis of fibrotic repair. Fibroblasts from fibrotic lungs of humans and mice demonstrate resistance to apoptosis induced by Fas-ligand and prior studies have shown that susceptibility to apoptosis is enhanced when Fas (CD95) expression is increased in these cells. Moreover, prior work shows that Fas expression in fibrotic lung fibroblasts is reduced by epigenetic silencing of the Fas promoter. However, the mechanisms by which microenvironmental stimuli such as TGF-?1 and substrate stiffness affect fibroblast Fas expression are not well understood. METHODS:Primary normal human lung fibroblasts (IMR-90) were cultured on tissue culture plastic or on polyacrylamide hydrogels with Young's moduli to recapitulate the compliance of normal (400 Pa) or fibrotic (6400 Pa) lung tissue and treated with or without TGF-?1 (10 ng/mL) in the presence or absence of protein kinase inhibitors and/or inflammatory cytokines. Expression of Fas was assessed by quantitative real time RT-PCR, ELISA and Western blotting. Soluble Fas (sFas) was measured in conditioned media by ELISA. Apoptosis was assessed using the Cell Death Detection Kit and by Western blotting for cleaved PARP. RESULTS:Fas expression and susceptibility to apoptosis was diminished in fibroblasts cultured on 6400 Pa substrates compared to 400 Pa substrates. TGF-?1 reduced Fas mRNA and protein in a time- and dose-dependent manner dependent on focal adhesion kinase (FAK). Surprisingly, TGF-?1 did not significantly alter cell-surface Fas expression, but did stimulate secretion of sFas. Finally, enhanced Fas expression and increased susceptibility to apoptosis was induced by combined treatment with TNF-?/IFN-? and was not inhibited by TGF-?1. CONCLUSIONS:Soluble and matrix-mediated pro-fibrotic stimuli promote fibroblast resistance to apoptosis by decreasing Fas transcription while stimulating soluble Fas secretion. These findings suggest that distinct mechanisms regulating Fas expression in fibroblasts may serve different functions in the complex temporal and spatial evolution of normal and fibrotic wound-repair responses.
| S-EPMC5946418 | biostudies-literature
Project description:We have previously shown that insulin-like growth factor-binding protein-5 (IGFBP-5) is overexpressed in fibrotic lung tissues and that it induces production of extracellular matrix components such as collagen and fibronectin both in vitro and in vivo. We recently observed mononuclear cell infiltration in lung tissues of mice expressing IGFBP-5. We therefore examined the role of IGFBP-5 on the migration of immune cells. Migration assays demonstrated that IGFBP-5 induced migration of peripheral blood mononuclear cells (PBMCs) in a dose-dependent manner. Preferential migration of monocytes/macrophages, natural killer cells, and T cells was observed. Moreover, the CD4/CD8 ratio of migrating cells was significantly higher in vitro and in vivo in response to IGFBP-5. IGFBP-5 resulted in preferential migration of activated CD4(+) T cells and monocytes. Interestingly, IGFBP-5 also induced migration of primary human lung fibroblasts. Exogenous administration of IGFBP-5 induced activation of mitogen-activated protein kinase (MAPK) signaling cascade but not PI3K in PBMCs. IGFBP-5-induced migration was blocked by the MEK1/2 inhibitor U0126, suggesting that IGFBP-5-induced migration occurs via MAPK activation. Furthermore, monocytes treated with recombinant IGFBP-5 expressed the mesenchymal markers alpha-smooth muscle actin and fibronectin in vitro and in vivo, suggesting that IGFBP-5 can induce the transformation of monocytes into mesenchymal cells. Collectively, our results suggest that IGFBP-5 induces cell migration via MAPK-dependent and IGF-I-independent mechanisms.
| S-EPMC2715907 | biostudies-other
Project description:Intermittent hypoxia (IH) is the predominant pathophysiological disturbance in obstructive sleep apnea (OSA), known to be independently associated with cardiovascular diseases. However, the effect of IH on cardiac fibrosis and molecular events involved in this process are unclear. Here, we tested IH in angiotensin II (Ang II)-induced cardiac fibrosis and signaling linked to fibroblast activation. IH triggered cardiac fibrosis and aggravated Ang II-induced cardiac dysfunction in mice. Plasma thrombospondin-1 (TSP1) content was upregulated in both IH-exposed mice and OSA patients. Moreover, both in vivo and in vitro results showed IH-induced cardiac fibroblast activation and increased TSP1 expression in cardiac fibroblasts. Mechanistically, phosphorylation of STAT3 at Tyr705 mediated the IH-induced TSP1 expression and fibroblast activation. Finally, STAT3 inhibitor S3I-201 or AAV9 carrying a periostin promoter driving the expression of shRNA targeting Stat3 significantly attenuated the synergistic effects of IH and Ang II on cardiac fibrosis in mice. This work suggests a potential therapeutic strategy for OSA-related fibrotic heart disease.
| S-EPMC6992386 | biostudies-literature
Project description:Idiopathic pulmonary fibrosis (IPF) is a life-threatening disease with limited treatment options. Additionally, the lack of a complete understanding of underlying immunological mechanisms underscores the importance of discovering novel options for therapeutic intervention. Since the PI3K/PTEN pathway in myeloid cells influences their effector functions, we wanted to elucidate how sustained PI3K activity induced by cell-type specific genetic deficiency of its antagonist PTEN modulates IPF, in a murine model of bleomycin-induced pulmonary fibrosis (BIPF). We found that myeloid PTEN deficient mice (PTEN(MyKO)), after induction of BIPF, exhibit increased TGF-?1 activation, mRNA expression of pro-collagens and lysyl oxidase as well as augmented collagen deposition compared to wild-type littermates, leading to enhanced morbidity and decreased survival. Analysis of alveolar lavage and lung cell composition revealed that PTEN(MyKO) mice exhibit reduced numbers of macrophages and T-cells in response to bleomycin, indicating an impaired recruitment function. Interestingly, we found dysregulated macrophage polarization as well as elevated expression and release of the pro-fibrotic cytokines IL-6 and TNF-? in PTEN(MyKO) mice during BIPF. This might point to an uncontrolled wound healing response in which the inflammatory as well as tissue repair mechanisms proceed in parallel, thereby preventing resolution and at the same time promoting extensive fibrosis.
| S-EPMC4789787 | biostudies-literature
Project description:Radiotherapy, a common component in cancer treatment, can induce adverse effects including fibrosis in co-irradiated tissues. We previously showed that differential DNA methylation at an enhancer of diacylglycerol kinase alpha (DGKA) in normal dermal fibroblasts is associated with radiation-induced fibrosis. After irradiation, the transcription factor EGR1 is induced and binds to the hypomethylated enhancer, leading to increased DGKA and pro-fibrotic marker expression. We now modulated this DGKA induction by targeted epigenomic and genomic editing of the DGKA enhancer and administering epigenetic drugs. Targeted DNA demethylation of the DGKA enhancer in HEK293T cells resulted in enrichment of enhancer-related histone activation marks and radiation-induced DGKA expression. Mutations of the EGR1-binding motifs decreased radiation-induced DGKA expression in BJ fibroblasts and caused dysregulation of multiple fibrosis-related pathways. EZH2 inhibitors (GSK126, EPZ6438) did not change radiation-induced DGKA increase. Bromodomain inhibitors (CBP30, JQ1) suppressed radiation-induced DGKA and pro-fibrotic marker expression. Similar drug effects were observed in donor-derived fibroblasts with low DNA methylation. Overall, epigenomic manipulation of DGKA expression may offer novel options for a personalized treatment to prevent or attenuate radiotherapy-induced fibrosis.
| S-EPMC8158145 | biostudies-literature
Project description:Central nervous system myelination requires the synthesis of large amounts of myelin basic protein (MBP) at the axon-glia contact site. MBP messenger RNA (mRNA) is transported in RNA granules to oligodendroglial processes in a translationally silenced state. This process is regulated by the trans-acting factor heterogeneous nuclear ribonucleoprotein (hnRNP) A2 binding to the cis-acting A2 response element (A2RE). Release of this repression of MBP mRNA translation is thus essential for myelination. Mice deficient in the Src family tyrosine kinase Fyn are hypomyelinated and contain reduced levels of MBP. Here, we identify hnRNP A2 as a target of activated Fyn in oligodendrocytes. We show that active Fyn phosphorylates hnRNP A2 and stimulates translation of an MBP A2RE-containing reporter construct. Neuronal adhesion molecule L1 binding to oligodendrocytes results in Fyn activation, which leads to an increase in hnRNP A2 phosphorylation. These results suggest that Fyn kinase activation results in the localized translation of MBP mRNA at sites of axon-glia contact and myelin deposition.
| S-EPMC2386098 | biostudies-literature
Project description:ObjectiveInflammation and fibrosis are intertwined in multiple disease processes. We have previously found that over-expression of urokinase plasminogen activator in macrophages induces spontaneous macrophage accumulation and fibrosis specific to the heart in mice. Understanding the relationship between inflammation and fibrosis in the heart is critical to developing therapies for diverse myocardial diseases. Therefore, we sought to determine if uPA induces changes in macrophage function that promote cardiac collagen accumulation.Methods and resultsWe analyzed the effect of the uPA transgene on expression of pro-inflammatory (M1) and pro-fibrotic (M2) genes and proteins in hearts and isolated macrophages of uPA overexpressing mice. We found that although there was elevation of the pro-inflammatory cytokine IL-6 in hearts of transgenic mice, IL-6 is not a major effector of uPA induced cardiac fibrosis. However, uPA expressing bone marrow-derived macrophages are polarized to express M2 genes in response to IL-4 stimulation, and these M2 genes are upregulated in uPA expressing macrophages following migration to the heart. In addition, while uPA expressing macrophages express a transcriptional profile that is seen in tumor-associated macrophages, these macrophages promote collagen expression in cardiac but not embryonic fibroblasts.ConclusionsUrokinase plasminogen activator induces an M2/profibrotic phenotype in macrophages that is fully expressed after migration of macrophages into the heart. Understanding the mechanisms by which uPA modulates macrophage function may reveal insights into diverse pathologic processes.
| S-EPMC3594198 | biostudies-literature
Project description:In fibroblasts, TGFβ1 stimulates IL11 upregulation that leads to an autocrine loop of IL11-dependent pro-fibrotic protein translation. The signaling pathways downstream of IL11, which acts via IL6ST, are contentious with both STAT3 and ERK implicated. Here we dissect IL11 signaling in fibroblasts and study IL11-dependent protein synthesis pathways in the context of approved anti-fibrotic drug mechanisms of action. We show that IL11-induced ERK activation drives fibrogenesis and while STAT3 phosphorylation (pSTAT3) is also seen, this appears unrelated to fibroblast activation. Ironically, recombinant human IL11, which has been used extensively in mouse experiments to infer STAT3 activity downstream of IL11, increases pSTAT3 in Il11ra1 null mouse fibroblasts. Unexpectedly, inhibition of STAT3 was found to induce severe proteotoxic ER stress, generalized fibroblast dysfunction and cell death. In contrast, inhibition of ERK prevented fibroblast activation in the absence of ER stress. IL11 stimulated an axis of ERK/mTOR/P70RSK protein translation and its selectivity for Collagen 1 synthesis was ascribed to an EPRS-regulated, ribosome stalling mechanism. Surprisingly, the anti-fibrotic drug nintedanib caused dose-dependent ER stress and lesser pSTAT3 expression. Pirfenidone had no effect on ER stress whereas anti-IL11 specifically inhibited the ERK/mTOR axis while reducing ER stress. These studies define the translation-specific signaling pathways downstream of IL11, intersect immune and metabolic signaling and reveal unappreciated effects of nintedanib.
| S-EPMC8505966 | biostudies-literature