Hypoxia promotes osteogenesis by facilitating acetyl-CoA-mediated mitochondrial-nuclear communication.

Bone-derived mesenchymal stem cells (MSCs) reside in a hypoxic niche that maintains their differentiation potential. While hypoxia (low oxygen concentration) was reported to critically support stem cell function and osteogenesis, the molecular events triggering changes in stem cell fate decisions in response to normoxia (high oxygen concentration) remain elusive. Here, we study the impact of normoxia on mitochondrial-nuclear communication during stem cell differentiation. We show that normoxia-cultured murine MSCs undergo profound transcriptional alterations which cause irreversible osteogenesis defects. Mechanistically, high oxygen promotes chromatin compaction and histone hypo-acetylation, particularly on promoters and enhancers of osteogenic genes. Although normoxia induces metabolic rewiring resulting in elevated acetyl-CoA levels, histone hypo-acetylation occurs due to the trapping of acetyl-CoA inside mitochondria owing to decreased citrate carrier (CiC) activity. Restoring the cytosolic acetyl-CoA pool remodels the chromatin landscape and rescues the osteogenic defects. Collectively, our results demonstrate that the metabolism-chromatin-osteogenesis axis is perturbed upon exposure to high oxygen levels and identifies CiC as a novel, oxygen-sensitive regulator of the MSC function.

Chromatin remodeling due to degradation of citrate carrier impairs osteogenesis of aged mesenchymal stem cells.

Aging is accompanied by a general decline in the function of many cellular pathways. However, whether these are causally or functionally interconnected remains elusive. Here, we study the effect of mitochondrial-nuclear communication on stem cell aging. We show that aged mesenchymal stem cells exhibit reduced chromatin accessibility and lower histone acetylation, particularly on promoters and enhancers of osteogenic genes. The reduced histone acetylation is due to impaired export of mitochondrial acetyl-CoA, owing to the lower levels of citrate carrier (CiC). We demonstrate that aged cells showed enhanced lysosomal degradation of CiC, which is mediated via mitochondrial-derived vesicles. Strikingly, restoring cytosolic acetyl-CoA levels either by exogenous CiC expression or via acetate supplementation, remodels the chromatin landscape and rescues the osteogenesis defects of aged mesenchymal stem cells. Collectively, our results establish a tight, age-dependent connection between mitochondrial quality control, chromatin and stem cell fate, which are linked together by CiC.

The decrease in histone methyltransferase EZH2 in response to fluid shear stress alters endothelial gene expression and promotes quiescence.

High uniform fluid shear stress (FSS) is atheroprotective and preserves the endothelial phenotype and function through activation of downstream mediators such as MAPK7 (Erk5). Endothelial cells respond to FSS thanks to mechanotransduction. However, how the resulting signaling is integrated and resolved at the epigenetic level remains elusive. We hypothesized that Polycomb methyltransferase EZH2 is involved in the effects of FSS in human endothelial cells. We showed that FSS decreases the expression of the Polycomb methyltransferase EZH2. Despite simultaneous activation of MAPK7, MAPK7 pathway does not directly influence the transcription of EZH2. Interestingly though, the knockdown of EZH2 activates the protective MAPK7 signaling in endothelial cells, even in the absence of FSS. To understand the influence of the FSS-decreased expression of EZH2 on endothelial transcriptome, we performed RNA-seq and differential gene expression analysis. We identified candidate groups of genes dependent on both EZH2 and FSS. Among those, Gene Ontology overrepresentation analysis revealed highly significant enrichment of the cell cycle-related genes, suggesting changes in proliferation. Indeed, the depletion of EZH2 strongly inhibited endothelial proliferation, indicating cell cycle arrest. The concomitant decrease in CCNA expression suggests the transition of endothelial cells into a quiescent phenotype. Further bioinformatical analysis suggested TXNIP as a possible mediator between EZH2 and cell cycle-related gene network. Our data show that EZH2 is a FSS-responsive gene. Decreased EZH2 levels enhance the activation of the atheroprotective MAPK7 signaling. Decrease in EZH2 under FSS mediates the decrease in the expression of the network of cell cycle-related genes, which allows the cells to enter quiescence. EZH2 is therefore important for the protective effects of FSS in endothelium.

Nrf2-heme oxygenase-1 axis in mucoepidermoid carcinoma of the lung: Antitumoral effects associated with down-regulation of matrix metalloproteinases.

Lung mucoepidermoid carcinoma (MEC) is a very poorly characterized rare subtype of non-small-cell lung cancer (NSCLC) associated with more favorable prognoses than other forms of intrathoracic malignancies. We have previously identified that heme oxygenase-1 (HO-1, encoded by HMOX1) inhibits MEC tumor growth and modulates the transcriptome of microRNAs. Here we investigate the role of a major upstream regulator of HO-1 and a master regulator of cellular antioxidant responses, transcription factor Nrf2, in MEC biology. Nrf2 overexpression in the NCI-H292 MEC cell line mimicked the phenotype of HO-1 overexpressing cells, leading to inhibition of cell proliferation and migration and down-regulation of oncogenic miR-378. HMOX1 silencing identified HO-1 as a major mediator of Nrf2 action. Nrf2- and HO-1 overexpressing cells exhibited strongly diminished expression of multiple matrix metalloproteinases and inflammatory cytokine interleukin-1ß, which was confirmed in an NCI-HO-1 xenograft model. Overexpression of HO-1 altered not only human MMP levels in tumor cells but also murine MMP levels within tumor microenvironment and metastatic niche. This could possibly contribute to decreased metastasis to the lungs and inhibitory effects of HO-1 on MEC tumor growth. Our profound transcriptome analysis and molecular characterization of the mucoepidermoid lung carcinoma helps to understand the specific clinical presentations of these tumors, emphasizing a unique antitumoral role of the Nrf2-HO-1 axis.

Endothelial-to-mesenchymal transition contributes to fibro-proliferative vascular disease and is modulated by fluid shear stress.

AIMS: Neointimal hyperplasia is a common feature of fibro-proliferative vascular disease and characterizes initial stages of atherosclerosis. Neointimal lesions mainly comprise smooth muscle-like cells. The presence of these lesions is related to local differences in shear stress. Neointimal cells may arise through migration and proliferation of smooth muscle cells from the media. However, a role for the endothelium as a source of smooth muscle-like cells has largely been disregarded. Here, we investigated the role of endothelial-to-mesenchymal transition (EndMT) in neointimal hyperplasia and atherogenesis, and studied its modulation by shear stress. METHODS AND RESULTS: In human atherosclerotic plaques and porcine aortic tissues, myo-endothelial cells were identified, suggestive for EndMT. Flow disturbance by thoracic-aortic constriction in mice similarly showed the presence of myo-endothelial cells specifically in regions exposed to disturbed flow. While uniform laminar shear stress (LSS) was found to inhibit EndMT, endothelial cells exposed to disturbed flow underwent EndMT, in vitro and in vivo, and showed atherogenic differentiation. Gain- and loss-of-function studies using a constitutive active mutant of MEK5 and short hairpins targeting ERK5 established a pivotal role for ERK5 signalling in the inhibition of EndMT. CONCLUSION: Together, these data suggest that EndMT contributes to neointimal hyperplasia and induces atherogenic differentiation of endothelial cells. Importantly, we uncovered that EndMT is modulated by shear stress in an ERK5-dependent manner. These findings provide new insights in the role of adverse endothelial plasticity in vascular disease and identify a novel atheroprotective mechanism of uniform LSS, namely inhibition of EndMT.

Enhancer of zeste homolog-2 (EZH2) methyltransferase regulates transgelin/smooth muscle-22α expression in endothelial cells in response to interleukin-1ß and transforming growth factor-ß2.

Smooth muscle-22α (SM22α), encoded by transgelin (TAGLN), is expressed in mesenchymal lineage cells, including myofibroblasts and smooth muscle cells. It is an F-actin binding protein that regulates the organization of actin cytoskeleton, cellular contractility and motility. SM22α is crucial for the maintenance of smooth muscle cell phenotype and its function. SM22α is also expressed in the processes of mesenchymal transition of epithelial (EMT) or endothelial cells (EndMT). The expression of TAGLN/SM22α is induced by transforming growth factor-ß (TGFß) signaling and enhanced by concomitant interleukin-1ß (IL-1ß) signaling. We investigated the epigenetic regulation of TAGLN expression by enhancer of zeste homolog-2 (EZH2), the methyltransferase of Polycomb, in the context of TGFß and IL-1ß signaling in endothelial cells. We demonstrate that the expression of EZH2 in endothelial cells was regulated by the inflammatory cytokine IL-1ß. A decrease in both expression and activity of EZH2 led to an increase in TAGLN expression. Inhibition of EZH2 augmented TGFß2-induced SM22α expression. The decrease of EZH2 levels in endothelial cells co-stimulated with IL-1ß and TGFß2 correlated with decreased H3K27me3 levels at the TAGLN proximal promoter. Moreover, the SM22α expression increased. Taken together, this suggests that EZH2 regulates the chromatin structure at the TAGLN promoter through tri-methylation of H3K27. EZH2 therefore acts as an epigenetic integrator of IL-1ß and TGFß2 signaling, providing an example of how cellular signaling can be resolved at the level of epigenetic regulation. Since IL-1ß and TGFß2 represent the pro-inflammatory and pro-fibrotic conditions during vascular fibroproliferative disease, we surmise that EZH2, as the molecule that integrates their signaling, could also be a promising target for development of future therapy.

Nrf2 regulates angiogenesis: effect on endothelial cells, bone marrow-derived proangiogenic cells and hind limb ischemia.

AIMS: Nuclear factor E2-related factor 2 (Nrf2), a key cytoprotective transcription factor, regulates also proangiogenic mediators, interleukin-8 and heme oxygenase-1 (HO-1). However, hitherto its role in blood vessel formation was modestly examined. Particularly, although Nrf2 was shown to affect hematopoietic stem cells, it was not tested in bone marrow-derived proangiogenic cells (PACs). Here we investigated angiogenic properties of Nrf2 in PACs, endothelial cells, and inflammation-related revascularization. RESULTS: Treatment of endothelial cells with angiogenic cytokines increased nuclear localization of Nrf2 and induced expression of HO-1. Nrf2 activation stimulated a tube network formation, while its inhibition decreased angiogenic response of human endothelial cells, the latter effect reversed by overexpression of HO-1. Moreover, lack of Nrf2 attenuated survival, proliferation, migration, and angiogenic potential of murine PACs and affected angiogenic transcriptome in vitro. Additionally, angiogenic capacity of PAC Nrf2(-/-) in in vivo Matrigel assay and PAC mobilization in response to hind limb ischemia of Nrf2(-/-) mice were impaired. Despite that, restoration of blood flow in Nrf2-deficient ischemic muscles was better and accompanied by increased oxidative stress and inflammatory response. Accordingly, the anti-inflammatory agent etodolac tended to diminish blood flow in the Nrf2(-/-) mice. INNOVATION: Identification of a novel role of Nrf2 in angiogenic signaling of endothelial cells and PACs. CONCLUSION: Nrf2 contributes to angiogenic potential of both endothelial cells and PACs; however, its deficiency increases muscle blood flow under tissue ischemia. This might suggest a proangiogenic role of inflammation in the absence of Nrf2 in vivo, concomitantly undermining the role of PACs in such conditions.

IL-1ß and TGFß2 synergistically induce endothelial to mesenchymal transition in an NFκB-dependent manner.

Endothelial to mesenchymal transition (EndMT) contributes to fibrotic diseases. The main inducer of EndMT is TGFß signaling. TGFß2 is the dominant isoform in the physiological embryonic EndMT, but its role in the pathological EndMT in the context of inflammatory co-stimulation is not known. The aim of this study was to investigate TGFß2-induced EndMT in the context of inflammatory IL-1ß signaling. Co-stimulation with IL-1ß and TGFß2, but not TGFß1, caused synergistic induction of EndMT. Also, TGFß2 was the only TGFß isoform that was progressively upregulated during EndMT. External IL-1ß stimulation was dispensable once EndMT was induced. The inflammatory transcription factor NFκB was upregulated in an additive manner by IL-1ß and TGFß2 co-stimulation. Co-stimulation also led to the nuclear translocation of NFκB which was sustained over long-term treatment. Activation of NFκB was indispensable for the co-induction of EndMT. Our data suggest that the microenvironment at the verge between inflammation (IL-1ß) and tissue remodeling (TGFß2) can strongly promote the process of EndMT. Therefore our findings provide new insights into the mechanisms of pathological EndMT.