Pulmonary hypertension (PH) is a multifaceted vascular disease where development and severity are determined by both genetic and environmental factors. Over the past decade, there has been an ...acceleration of the discovery of molecular effectors that mediate PH pathogenesis, including large numbers of microRNA molecules that are expressed in pulmonary vascular cell types and exert system-wide regulatory functions in all aspects of vascular health and disease. Due to the inherent pleiotropy, overlap, and redundancy of these molecules, it has been challenging to define their integrated effects on overall disease manifestation. In this review, we summarize our current understanding of the roles of microRNAs in PH with an emphasis on potential methods to discern the hierarchical motifs governing their multifunctional and interconnected activities. Deciphering this higher order of regulatory structure will be crucial for overcoming the challenges of developing these molecules as biomarkers or therapeutic targets, in isolation or combination.
TEAD1 and the mammalian Hippo pathway regulate cellular proliferation and function, though their regulatory function in β cells remains poorly characterized. In this study, we demonstrate that while ...β cell-specific TEAD1 deletion results in a cell-autonomous increase of β cell proliferation, β cell-specific deletion of its canonical coactivators, YAP and TAZ, does not affect proliferation, suggesting the involvement of other cofactors. Using an improved split-GFP system and yeast two-hybrid platform, we identify VGLL4 and MENIN as TEAD1 corepressors in β cells. We show that VGLL4 and MENIN bind to TEAD1 and repress the expression of target genes, including FZD7 and CCN2, which leads to an inhibition of β cell proliferation. In conclusion, we demonstrate that TEAD1 plays a critical role in β cell proliferation and identify VGLL4 and MENIN as TEAD1 corepressors in β cells. We propose that these could be targeted to augment proliferation in β cells for reversing diabetes.
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•YAP and TAZ do not have a physiological role in β cell function and proliferation•VGLL4 and MENIN are important repressors of TEAD1 activity in β cells•TEAD1 inhibits its own expression in an auto-feedback loop•Improved split-GFP (iSG) system is a sensitive assay for unknown protein interactions
Feng et al. report that YAP and TAZ do not have a physiological role in β cell proliferation and function. They identify VGLL4 and MENIN as TEAD1 corepressors in regulating β cell proliferation and propose that these may be targeted to augment proliferation in β cells for reversing diabetes.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Idiopathic pulmonary fibrosis (IPF) is a fatal disorder resulting from the progressive remodeling of lungs, with no known effective treatment. Although transforming growth factor (TGF)-β has a ...well-established role in lung fibrosis, clinical experience with neutralizing antibodies to TGF-β has been disappointing, and strategies to directly suppress TGF-β1 secretion are needed. In this study we used a combination of in silico, in vitro, and in vivo approaches to identify microRNAs involved in TGF-β1 regulation and to validate the role of miR-326 in pulmonary fibrosis.We show that hsa-miR-326 regulates TGF-β1 expression and that hsa-miR-326 levels are inversely correlated to TGF-β1 protein levels in multiple human cell lines. The increase in TGF-β1 expression during the progression of bleomycin-induced lung fibrosis in mice was associated with loss of mmu-miR-326. Restoration of mmu-miR-326 levels by intranasal delivery of miR-326 mimics was sufficient to inhibit TGF-β1 expression and attenuate the fibrotic response. Moreover, human IPF lung specimens had markedly diminished miR-326 expression as compared with nonfibrotic lungs. Additional targets of miR-326 controlling TGF-β signaling and fibrosis-related pathways were identified, and miR-326 was found to down-regulate profibrotic genes, such as Ets1, Smad3, and matrix metalloproteinase 9, whereas it up-regulates antifibrotic genes, such as Smad7. Our results suggest for the first time that miR-326 plays a key role in regulating TGF-β1 expression and other profibrotic genes and could be useful in developing better therapeutic strategies for alleviating lung fibrosis.
The Hippo-TEAD pathway regulates cellular proliferation and function. The existing paradigm is that TEAD co-activators, YAP and TAZ, and co-repressor, VGLL4, bind to the pocket region of TEAD1 to ...enable transcriptional activation or repressive function. Here we demonstrate a pocket-independent transcription repression mechanism whereby TEAD1 controls cell proliferation in both non-malignant mature differentiated cells and in malignant cell models. TEAD1 overexpression can repress tumor cell proliferation in distinct cancer cell lines. In pancreatic β cells, conditional knockout of TEAD1 led to a cell-autonomous increase in proliferation. Genome-wide analysis of TEAD1 functional targets via transcriptomic profiling and cistromic analysis revealed distinct modes of target genes, with one class of targets directly repressed by TEAD1. We further demonstrate that TEAD1 controls target gene transcription in a motif-dependent and orientation-independent manner. Mechanistically, we show that TEAD1 has a pocket region-independent, direct repressive function via interfering with RNA polymerase II (POLII) binding to target promoters. Our study reveals that TEAD1 target genes constitute a mutually restricted regulatory loop to control cell proliferation and uncovers a novel direct repression mechanism involved in its transcriptional control that could be leveraged in future studies to modulate cell proliferation in tumors and potentially enhance the proliferation of normal mature cells.
RNA editing of miRNAs, especially in the seed region, adds another layer to miRNA mediated gene regulation which can modify its targets, altering cellular signaling involved in important processes ...such as differentiation. In this study, we have explored the role of miRNA editing in CD4(+) T cell differentiation. CD4(+) T cells are an integral component of the adaptive immune system. Naïve CD4(+) T cells, on encountering an antigen, get differentiated either into inflammatory subtypes like Th1, Th2 or Th17, or into immunosuppressive subtype Treg, depending on the cytokine milieu. We found C-to-U editing at fifth position of mature miR-100, specifically in Treg. The C-to-U editing of miR-100 is functionally associated with at least one biologically relevant target change, from MTOR to SMAD2. Treg cell polarization by TGFβ1 was reduced by both edited and unedited miR-100 mimics, but percentage of Treg in PBMCs was only reduced by edited miR-100 mimics, suggesting a model in which de-repression of MTOR due to loss of unedited mir-100, promotes tolerogenic signaling, while gain of edited miR-100 represses SMAD2, thereby limiting the Treg. Such delicately counterbalanced systems are a hallmark of immune plasticity and we propose that miR-100 editing is a novel mechanism toward this end.
The transcriptional factor, Tead1, mediates the transcriptional output of the Hippo pathway. Tead1 activates the transcription of downstream genes including many cell cycle promoting genes that lead ...to cell proliferation in many tissues, but its role in β-cells is unknown. To test if Tead1 is required for β-cell proliferation and function, we deleted Tead1 in β-cell. Deletion of Tead1 in β-cells was confirmed in these β-cell Tead1 KO (β-Tead1-/-) , by a significant decrease in transcript and protein from whole islet lysates. The β-Tead1-/- mice had normal body weight but developed significantly higher fasting and fed glucose levels starting at 5 weeks of age and progressed to frank diabetes by 8 weeks, with fasting blood glucose >300 mg/dl, accompanied by hypoinsulinemia, as compared to floxed control mice. 8wk β-Tead1-/- mice display severe glucose intolerance due to an abrogation of GSIS during GTT, with a loss of first phase insulin secretion. Pancreatic insulin content was decreased by ∼50%, due to a decrease in expression of mature β-cell genes including, Pdx1, Nkx6.1 and MafA, all of which, we show to be direct transcriptional targets of Tead1. Tead1 also binds to the proximal promoter at the Ink4a locus to regulate p16/p19, critical cell cycle inhibitors, and this loss of activation in Tead1-deficient β-cells leads to an enhanced entry into cell cycle. Furthermore, to test if Tead1 has a similar regulatory role in human β-cells, we deleted TEAD1 knockout in human iPS cell-derived β-cells using CRISPR/Cas9 system. Tead1 deficient iPS cell-derived β-cells displayed a significant decrease in vitro GSIS. Our results demonstrate Tead1 to be a transcriptional switch required for β-cells to maintain mature function and remain in proliferative quiescence. This novel regulatory pathway could be targeted for β-cell replacement therapy to achieve β-cell proliferation without a loss of mature function.
Disclosure
J. Lee: None. V. Yechoor: n/a. R. Liu: None. F. Li: None. V. Negi: None. R. Jagannathan: None. M. Huising: Consultant; Crinetics Pharmaceuticals, Inc., Research Support; Crinetics Pharmaceuticals, Inc. K. Ma: None. B. Shih: None. M. Moulik: None.
Funding
VA-MERIT Grant (I01BX002678)
β-cell mass is significantly reduced especially in the late stage of diabetes. Hence, increasing β-cell proliferation to increase β-cell mass remains a desirable goal for diabetes treatment. The ...mechanisms that regulate β cell proliferation and concurrent maintenance of mature function is not well understood. Hippo-TEAD pathways regulated proliferation in many tissues. Our data show that TEAD1 (Hippo) pathway activities were significantly reduced in mature β cell. TEAD1 knockout results in the cell-autonomous increase of β cell proliferation, while loss of TAZ has not change in β cell proliferation, suggesting that TEAD1 corepressors, rather than coactivators, play a dominant role in β-cells. Improved Split-GFP system and yeast two-hybrid platform were also adapted to screen TEAD1 cofactors in β cells. Our study showed that MENIN can bind to TEAD1 directly and the pocket area of TEAD1 is critical for this binding. VGLL4 and MENIN work as TEAD1 corepressors to inhibit the expression of target genes in β-cells, including FZD7 and CCN2, which leads to a restriction of β cell proliferation. We also found that TEAD1 can regulate TEAD1, and this auto-inhibition contributes to TEAD1 pathway inhibition with β cell maturity. Our study sheds light on the mechanisms involved in β cell maintenance of mature function and proliferation and offer the mechanistic basis for modulating TEAD pathway to increase proliferation in β cells.
Disclosure
F.Li: None. R.Liu: None. V.Negi: None. P.Yang: None. J.K.Lee: None. M.Moulik: None. V.Yechoor: n/a.
Abstract only Background: The pathogenic mechanisms underlying HFpEF (Heart Failure with preserved Ejection Fraction), which accounts for 50% of heart failure, remain unclear. Elevated endoplasmic ...reticulum (ER) stress has been implicated in HFpEF. Circadian disruption (CD) as seen in shift workers has been associated with an excess risk for chronic age-related disorders, including cardiovascular disease. Currently, the role of CD in HFpEF is not known. Hypothesis: The dual 'hits' of chronic metabolic stress and circadian disruption lead to increased ER stress, impaired unfolded protein response (UPR), and adverse cardiac remodeling resulting in HFpEF. Methods: High fat diet (HFD) fed C57BL6 male mice were subjected to chronic shifted light-dark (LD) cycles mimicking shift work (Shift), with appropriate controls. Metabolic (glucose tolerance, insulin sensitivity, body fat) and cardiac function parameters (systolic and diastolic function by ECHO, MRI, and terminal PV-loops) were collected. Cardiac tissue was analyzed using qPCR, western blot, bulk RNA-seq, spatial transcriptomics, proteomics, and histology. For in vitro studies, Bmal1 was deleted in H9C2 cells using CRISPR/Cas9. Results: ‘HS’ mice (HFD & Shift) had the most insulin resistance, glucose intolerance, and obesity, compared to ‘HR’ (HFD & Regular LD), ‘CS’ (Normal Chow & Shift) and ‘CR’ (Chow and Regular LD) groups. ECHO and MRI revealed preserved EF in all groups, with diastolic impairment in both HS and HR (↑MV E/E’, ↓MV E/A, and ↓LV diastolic strain) groups, HS>HR. Only ‘HS’ mice showed ↓cardiac compliance (↑β in EDPVR) and ↑lung weight suggestive of HF consistent with HFpEF. Gene and protein expression revealed dysregulation of numerous UPR pathway molecules (Atf6, Chop, Bip, Perk, and others) in HS mice. Mechanistically, Bmal1 deletion in CMs resulted in similar dysregulation of UPR genes. HS mouse hearts showed increased fibrosis, and spatial transcriptomics displayed enrichment of ATF4-related pathways in CMs and activated fibroblasts. Conclusions: The dual hit of metabolic and circadian stress induces HFpEF, which cannot be recapitulated by only one stressor. Our results identify previously unrecognized roles of UPR perturbation in driving HFpEF under circadian and metabolic stress.
The COVID-pandemic has contributed to more than 5 million deaths worldwide in the last two years. Co-morbid conditions such as Type 2 Diabetes (T2D) , HTN, obesity, and CKD have been associated with ...increased mortality with COVID-19. In a large meta-analysis, the relative risk of mortality was 1.54 for patients with T2D and COVID-19. Thus, there is an imperative need to develop a platform for rapid and reliable drug screening/selection against COVID-related morbidity/mortality in T2D patients. With limited translatability of in vitro and small animal models to humans, human organ-on-a-chip models are an attractive platform to model in vivo disease conditions and test potential therapeutics. We seeded T2D or nondiabetes patient-derived macrophage and human liver sinusoidal endothelial cells along with normal hepatocytes and kupffer cells in the liver-on-a-chip (LAMPS - Liver Acinus MicroPhysiological System) developed by our group, perfused with media mimicking normal fasting or late metabolic syndrome (LMS - high levels of glucose, fatty acids, insulin, glucagon) states. We transduced both macrophage and endothelial cells to overexpress the SARS-CoV2-S (spike) protein and compared it with a control lentivirus transduction. We found that T2D cells overexpressing S-protein in LMS media (T2D chip) displayed an increased secretion of inflammatory cytokines compared to the nondiabetes chip over days. We then tested the effect of Tocilizumab (IL6-receptor antagonist) in T2D chips. Compared to vehicle control, Tocilizumab significantly decreased the S-protein induced inflammatory cytokine secretion in T2D chips but not in nondiabetes chips, indicating its higher efficacy in severe disease states only. This is consistent with what was observed in large clinical trials providing confirmatory evidence that the LAMPS T2D and nondiabetes chips serve as a relevant in vitro model system to replicate human in vivo pathophysiology of COVID and for screening potential therapeutics.
Disclosure
V.Negi: None. D.Gavlock: None. J.Lee: None. T.Shun: None. A.Gough: None. M.T.Miedel: None. D.L.Taylor: None. V.Yechoor: n/a.
Idiopathic Pulmonary Fibrosis (IPF) is a progressively fatal and incurable disease characterized by the loss of alveolar structures, increased epithelial–mesenchymal transition (EMT), and aberrant ...tissue repair. In this study, we investigated the role of Nuclear Factor I-B (NFIB), a transcription factor critical for lung development and maturation, in IPF. Using both human lung tissue samples from patients with IPF, and a mouse model of lung fibrosis induced by bleomycin, we showed that there was a significant reduction of NFIB both in the lungs of patients and mice with IPF. Furthermore, our in vitro experiments using cultured human lung cells demonstrated that the loss of NFIB was associated with the induction of EMT by transforming growth factor beta (TGF-β). Knockdown of NFIB promoted EMT, while overexpression of NFIB suppressed EMT and attenuated the severity of bleomycin-induced lung fibrosis in mice. Mechanistically, we identified post-translational regulation of NFIB by miR-326, a miRNA with anti-fibrotic effects that is diminished in IPF. Specifically, we showed that miR-326 stabilized and increased the expression of NFIB through its 3'UTR target sites for Human antigen R (HuR). Moreover, treatment of mice with either NFIB plasmid or miR-326 reversed airway collagen deposition and fibrosis. In conclusion, our study emphasizes the critical role of NFIB in lung development and maturation, and its reduction in IPF leading to EMT and loss of alveolar structures. Our study highlights the potential of miR-326 as a therapeutic intervention for IPF.
Graphical abstract
The schema shows the role of NFIB in maintaining the normal epithelial cell characteristics in the lungs and how its reduction leads to a shift towards mesenchymal cell-like features and pulmonary fibrosis.
A
In normal lungs, NFIB is expressed abundantly in the epithelial cells, which helps in maintaining their shape, cell polarity and adhesion molecules. However, when the lungs are exposed to factors that induce pulmonary fibrosis, such as bleomycin, or TGF-β, the epithelial cells undergo epithelial to mesenchymal transition (EMT), which leads to a decrease in NFIB.
B
The mesenchymal cells that arise from EMT appear as spindle-shaped with loss of cell junctions, increased cell migration, loss of polarity and expression of markers associated with mesenchymal cells/fibroblasts.
C
We designed a therapeutic approach that involves exogenous administration of NFIB in the form of overexpression plasmid or microRNA-326. This therapeutic approach decreases the mesenchymal cell phenotype and restores the epithelial cell phenotype, thus preventing the development or progression of pulmonary fibrosis.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
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