Integrated Stress Response Potentiates Ponatinib-Induced Cardiotoxicity.

BACKGROUND: Mitochondrial dysfunction is a primary driver of cardiac contractile failure; yet, the cross talk between mitochondrial energetics and signaling regulation remains obscure. Ponatinib, a tyrosine kinase inhibitor used to treat chronic myeloid leukemia, is among the most cardiotoxic tyrosine kinase inhibitors and causes mitochondrial dysfunction. Whether ponatinib-induced mitochondrial dysfunction triggers the integrated stress response (ISR) to induce ponatinib-induced cardiotoxicity remains to be determined. METHODS: Using human induced pluripotent stem cells-derived cardiomyocytes and a recently developed mouse model of ponatinib-induced cardiotoxicity, we performed proteomic analysis, molecular and biochemical assays to investigate the relationship between ponatinib-induced mitochondrial stress and ISR and their role in promoting ponatinib-induced cardiotoxicity. RESULTS: Proteomic analysis revealed that ponatinib activated the ISR in cardiac cells. We identified GCN2 (general control nonderepressible 2) as the eIF2α (eukaryotic translation initiation factor 2α) kinase responsible for relaying mitochondrial stress signals to trigger the primary ISR effector-ATF4 (activating transcription factor 4), upon ponatinib exposure. Mechanistically, ponatinib treatment exerted inhibitory effects on ATP synthase activity and reduced its expression levels resulting in ATP deficits. Perturbed mitochondrial function resulting in ATP deficits then acts as a trigger of GCN2-mediated ISR activation, effects that were negated by nicotinamide mononucleotide, an NAD+ precursor, supplementation. Genetic inhibition of ATP synthase also activated GCN2. Interestingly, we showed that the decreased abundance of ATP also facilitated direct binding of ponatinib to GCN2, unexpectedly causing its activation most likely because of a conformational change in its structure. Importantly, administering an ISR inhibitor protected human induced pluripotent stem cell-derived cardiomyocytes against ponatinib. Ponatinib-treated mice also exhibited reduced cardiac function, effects that were attenuated upon systemic ISRIB administration. Importantly, ISRIB does not affect the antitumor effects of ponatinib in vitro. CONCLUSIONS: Neutralizing ISR hyperactivation could prevent or reverse ponatinib-induced cardiotoxicity. The findings that compromised ATP production potentiates GCN2-mediated ISR activation have broad implications across various cardiac diseases. Our results also highlight an unanticipated role of ponatinib in causing direct activation of a kinase target despite its role as an ATP-competitive kinase inhibitor.

Blue LED causes autophagic cell death in human osteosarcoma by increasing ROS generation and dephosphorylating EGFR.

Osteosarcoma (OS) is the most common primary malignant bone tumour in adolescence. Lately, light-emitting diodes (LED)-based therapy has emerged as a new promising approach for several diseases. However, it remains unknown in human OS. Here, we found that the blue LED irradiation significantly suppressed the proliferation, migration and invasion of human OS cells, while we observed blue LED irradiation increased ROS production through increased NADPH oxidase enzymes NOX2 and NOX4, as well as decreased Catalase (CAT) expression levels. Furthermore, we revealed blue LED irradiation-induced autophagy characterized by alterations in autophagy protein markers including Beclin-1, LC3-II/LC3-I and P62. Moreover, we demonstrated an enhanced autophagic flux. The blockage of autophagy displayed a remarkable attenuation of anti-tumour activities of blue LED irradiation. Next, ROS scavenger N-acetyl-L-cysteine (NAC) and NOX inhibitor diphenyleneiodonium (DPI) blocked suppression of OS cell growth, indicating that ROS accumulation might play an essential role in blue LED-induced autophagic OS cell death. Additionally, we observed blue LED irradiation decreased EGFR activation (phosphorylation), which in turn led to Beclin-1 release and subsequent autophagy activation in OS cells. Analysis of EGFR colocalization with Beclin-1 and EGFR-immunoprecipitation (IP) assay further revealed the decreased interaction of EGFR and Beclin-1 upon blue LED irradiation in OS cells. In addition, Beclin-1 down-regulation abolished the effects of blue LED irradiation on OS cells. Collectively, we concluded that blue LED irradiation exhibited anti-tumour effects on OS by triggering ROS and EGFR/Beclin-1-mediated autophagy signalling pathway, representing a potential approach for human OS treatment.

YAP/TEAD3 signal mediates cardiac lineage commitment of human-induced pluripotent stem cells.

Cardiomyocytes differentiated from human-induced pluripotent stem cells (hiPSCs) hold great potential for therapy of heart diseases. However, the underlying mechanisms of its cardiac differentiation have not been fully elucidated. Hippo-YAP signal pathway plays important roles in cell differentiation, tissue homeostasis, and organ size. Here, we identify the role of Hippo-YAP signal pathway in determining cardiac differentiation fate of hiPSCs. We found that cardiac differentiation of hiPSCs were significantly inhibited after treatment with verteporfin (a selective and potent YAP inhibitor). During hiPSCs differentiation from mesoderm cells (MESs) into cardiomyocytes, verteporfin treatment caused the cells retained in the earlier cardiovascular progenitor cells (CVPCs) stage. Interestingly, during hiPSCs differentiation from CVPC into cardiomyocytes, verteporfin treatment induced cells dedifferentiation into the earlier CVPC stage. Mechanistically, we found that YAP interacted with transcriptional enhanced associate domain transcription factor 3 (TEAD3) to regulate cardiac differentiation of hiPSCs during the CVPC stage. Consistently, RNAi-based silencing of TEAD3 mimicked the phenotype as the cells treated with verteporfin. Collectively, our study suggests that YAP-TEAD3 signaling is important for cardiomyocyte differentiation of hiPSCs. Our findings provide new insight into the function of Hippo-YAP signal in cardiovascular lineage commitment.

Iron Homeostasis Determines Fate of Human Pluripotent Stem Cells Via Glycerophospholipids-Epigenetic Circuit.

Iron homeostasis is crucial for a variety of biological processes, but the biological role of iron homeostasis in pluripotent stem cells (PSCs) remains largely unknown. The present study aimed to determine whether iron homeostasis is involved in maintaining the pluripotency of human PSCs (hPSCs). We found that the intracellular depletion of iron leads to a rapid downregulation of NANOG and a dramatic decrease in the self-renewal of hPSCs as well as spontaneous and nonspecific differentiation. Moreover, long-term depletion of iron can result in the remarkable cell death of hPSCs via apoptosis and necrosis pathways. Additionally, we found that the depletion of iron increased the activity of lipoprotein-associated phospholipase A2 (LP-PLA2) and the production of lysophosphatidylcholine, thereby suppressing NANOG expression by enhancer of zeste homolog 2-mediated trimethylation of histone H3 lysine 27. Consistently, LP-PLA2 inhibition abrogated iron depletion-induced loss of pluripotency and differentiation. Altogether, the findings of our study demonstrates that iron homeostasis, acting through glycerophospholipid metabolic pathway, is essential for the pluripotency and survival of hPSCs. Stem Cells 2019;37:489-503.

The Long Non-coding RNA-ORLNC1 Regulates Bone Mass by Directing Mesenchymal Stem Cell Fate.

Bone marrow-derived mesenchymal stem cells (BMSCs) have the potential to differentiate into osteoblasts or adipocytes, and the shift between osteogenic and adipogenic differentiation determines bone mass. The aim of this study was to identify whether lncRNAs are involved in the differentiation commitment of BMSCs during osteoporosis. Here, we found ORLNC1, a functionally undefined lncRNA that is highly conserved, which exhibited markedly higher expression levels in BMSCs, bone tissue, and the serum of OVX-induced osteoporotic mice than sham-operated counterparts. Notably, a similar higher abundance of lncRNA-ORLNC1 expression was also observed in the bone tissue of osteoporotic patients. The transgenic mice overexpressing lncRNA-ORLNC1 showed a substantial increase in the osteoporosis-associated bone loss and decline in the osteogenesis of BMSCs. The BMSCs pretreated with lncRNA-ORLNC1-overexpressing lentivirus vector exhibited the suppressed capacity of osteogenic differentiation and oppositely enhanced adipogenic differentiation. We then established that lncRNA-ORLNC1 acted as a competitive endogenous RNA (ceRNA) for miR-296. Moreover, miR-296 was found markedly upregulated during osteoblast differentiation, and it accelerated osteogenic differentiation by targeting Pten. Taken together, our results indicated that the lncRNA-ORLNC1-miR-296-Pten axis may be a critical regulator of the osteoporosis-related switch between osteogenesis and adipogenesis of BMSCs and might represent a plausible therapeutic target for improving osteoporotic bone loss.

MicroRNA-92b-5p modulates melatonin-mediated osteogenic differentiation of bone marrow mesenchymal stem cells by targeting ICAM-1.

Osteoporosis is closely associated with the dysfunction of bone metabolism, which is caused by the imbalance between new bone formation and bone resorption. Osteogenic differentiation plays a vital role in maintaining the balance of bone microenvironment. The present study investigated whether melatonin participated in the osteogenic commitment of bone marrow mesenchymal stem cells (BMSCs) and further explored its underlying mechanisms. Our data showed that melatonin exhibited the capacity of regulating osteogenic differentiation of BMSCs, which was blocked by its membrane receptor inhibitor luzindole. Further study demonstrated that the expression of miR-92b-5p was up-regulated in BMSCs after administration of melatonin, and transfection of miR-92b-5p accelerated osteogenesis of BMSCs. In contrast, silence of miR-92b-5p inhibited the osteogenesis of BMSCs. The increase in osteoblast differentiation of BMSCs caused by melatonin was attenuated by miR-92b-5p AMO as well. Luciferase reporter assay, real-time qPCR analysis and western blot analysis confirmed that miR-92b-5p was involved in osteogenesis by directly targeting intracellular adhesion molecule-1 (ICAM-1). Melatonin improved the expression of miR-92b-5p, which could regulate the differentiation of BMSCs into osteoblasts by targeting ICAM-1. This study provided novel methods for treating osteoporosis.

Effects of Blue Light Emitting Diode Irradiation On the Proliferation, Apoptosis and Differentiation of Bone Marrow-Derived Mesenchymal Stem Cells.

BACKGROUND/AIMS: Blue light emitting diodes (LEDs) have been proven to affect the growth of several types of cells. The effects of blue LEDs have not been tested on bone marrow-derived mesenchymal stem cells (BMSCs), which are important for cell-based therapy in various medical fields. Therefore, the aim of this study was to determine the effects of blue LED on the proliferation, apoptosis and osteogenic differentiation of BMSCs. METHODS: BMSCs were irradiated with a blue LED light at 470 nm for 1 min, 5 min, 10 min, 30 min and 60 min or not irradiated. Cell proliferation was measured by performing cell counting and EdU staining assays. Cell apoptosis was detected by TUNEL staining. Osteogenic differentiation was evaluated by ALP and ARS staining. DCFH-DA staining and γ-H2A.X immunostaining were used to measure intracellular levels of ROS production and DNA damage. RESULTS: Both cell counting and EdU staining assays showed that cell proliferation of BMSCs was significantly reduced upon blue LED irradiation. Furthermore, treatment of BMSCs with LED irradiation was followed by a remarkable increase in apoptosis, indicating that blue LED light induced toxic effects on BMSCs. Likewise, BMSC osteogenic differentiation was inhibited after exposure to blue LED irradiation. Further, blue LED irradiation was followed by the accumulation of ROS production and DNA damage. CONCLUSIONS: Taken together, our study demonstrated that blue LED light inhibited cell proliferation, inhibited osteogenic differentiation, and induced apoptosis in BMSCs, which are associated with increased ROS production and DNA damage. These findings may provide important insights for the application of LEDs in future BMSC-based therapies.

Melatonin protects bone marrow mesenchymal stem cells against iron overload-induced aberrant differentiation and senescence.

Bone marrow mesenchymal stem cells (BMSCs) are an expandable population of stem cells which can differentiate into osteoblasts, chondrocytes and adipocytes. Dysfunction of BMSCs in response to pathological stimuli contributes to bone diseases. Melatonin, a hormone secreted from pineal gland, has been proved to be an important mediator in bone formation and mineralization. The aim of this study was to investigate whether melatonin protected against iron overload-induced dysfunction of BMSCs and its underlying mechanisms. Here, we found that iron overload induced by ferric ammonium citrate (FAC) caused irregularly morphological changes and markedly reduced the viability in BMSCs. Consistently, osteogenic differentiation of BMSCs was significantly inhibited by iron overload, but melatonin treatment rescued osteogenic differentiation of BMSCs. Furthermore, exposure to FAC led to the senescence in BMSCs, which was attenuated by melatonin as well. Meanwhile, melatonin was able to counter the reduction in cell proliferation by iron overload in BMSCs. In addition, protective effects of melatonin on iron overload-induced dysfunction of BMSCs were abolished by its inhibitor luzindole. Also, melatonin protected BMSCs against iron overload-induced ROS accumulation and membrane potential depolarization. Further study uncovered that melatonin inhibited the upregulation of p53, ERK and p38 protein expressions in BMSCs with iron overload. Collectively, melatonin plays a protective role in iron overload-induced osteogenic differentiation dysfunction and senescence through blocking ROS accumulation and p53/ERK/p38 activation.

Astragalus Polysaccharide Attenuated Iron Overload-Induced Dysfunction of Mesenchymal Stem Cells via Suppressing Mitochondrial ROS.

BACKGROUND/AIMS: Bone marrow-derived mesenchymal stem cells (BMSCs) have the ability to differentiate into multilineage cells such as osteoblasts, chondrocytes, and cardiomyocytes. Dysfunction of BMSCs in response to pathological stimuli participates in the development of diseases such as osteoporosis. Astragalus polysaccharide (APS) is a major active ingredient of Astragalus membranaceus, a commonly used anti-aging herb in traditional Chinese medicine. The aim of this study was to investigate whether APS protects against iron overload-induced dysfunction of BMSCs and its underlying mechanisms. METHODS: BMSCs were exposed to ferric ammonium citrate (FAC) with or without different concentrations of APS. The viability and proliferation of BMSCs were assessed by CCK-8 assay and EdU staining. Cell apoptosis, senescence and pluripotency were examined utilizing TUNEL staining, ß-galactosidase staining and qRT-PCR respectively. The reactive oxygen species (ROS) level was assessed in BMSCs with a DCFH-DA probe and MitoSOX Red staining. RESULTS: Firstly, we found that iron overload induced by FAC markedly reduced the viability and proliferation of BMSCs, but treatment with APS at 10, 30 and 100 µg/mL was able to counter the reduction of cell proliferation. Furthermore, exposure to FAC led to apoptosis and senescence in BMSCs, which were partially attenuated by APS. The pluripotent genes Nanog, Sox2 and Oct4 were shown to be downregulated in BMSCs after FAC treatment, however APS inhibited the reduction of Nanog, Sox2 and Oct4 expression. Further study uncovered that APS treatment abrogated the increase of intracellular and mitochondrial ROS level in FAC-treated BMSCs. CONCLUSION: Treatment of BMSCs with APS to impede mitochondrial ROS accumulation can remarkably inhibit apoptosis, senescence, and the reduction of proliferation and pluripotency of BMSCs caused by FAC-induced iron overload.

Long noncoding RNA H19 mediates melatonin inhibition of premature senescence of c-kit(+) cardiac progenitor cells by promoting miR-675.

Melatonin, a hormone secreted by the pineal gland, possesses multiple biological activities such as antitumor, antioxidant, and anti-ischemia. C-kit(+) cardiac progenitor cells (CPCs) have emerged as a promising tool for the treatment of heart diseases. However, the senescence of CPCs due to pathological stimuli leads to the decline of CPCs' functions and regenerative potential. This study was conducted to demonstrate whether melatonin antagonizes the senescence of CPCs in response to oxidative stress. Here, we found that the melatonin treatment markedly inhibited the senescent characteristics of CPCs after exposed to sublethal concentration of H2 O2 , including the increase in senescence-associated ß-galactosidase (SA-ß-gal)-positive CPCs, senescence-associated heterochromatin loci (SAHF), secretory IL-6 level, and the upregulation of p53 and p21 proteins. Senescence-associated proliferation reduction was also attenuated by melatonin in CPCs. Luzindole, the melatonin membrane receptor blocker, may block the melatonin-mediated suppression of premature senescence in CPCs. Interestingly, we found that long noncoding RNA H19 and its derived miR-675 were downregulated by H2 O2 in CPCs, but melatonin treatment could counter this alteration. Furthermore, knockdown of H19 or miR-675 blocked antisenescence actions of melatonin on H2 O2 -treated CPCs. It was further verified that H19-derived miR-675 targeted at the 3'UTR of USP10, which resulted in the downregulation of p53 and p21 proteins. In summary, melatonin antagonized premature senescence of CPCs via H19/miR-675/USP10 pathway, which provides new insights into pharmacological actions and potential applications of melatonin on the senescence of CPCs.

Estrogen counteracts age-related decline in beige adipogenesis through the NAMPT-regulated ER stress response.

Thermogenic beige adipocytes are recognized as potential therapeutic targets for combating metabolic diseases. However, the metabolic advantages that they offer are compromised with aging. Here we show that treating mice with estrogen (E2), a hormone that decreases with age, can counteract the age-related decline in beige adipogenesis when exposed to cold temperature while concurrently enhancing energy expenditure and improving glucose tolerance in mice. Mechanistically, we found that nicotinamide phosphoribosyl transferase (NAMPT) plays a pivotal role in facilitating the formation of E2-induced beige adipocytes, which subsequently suppresses the onset of age-related endoplasmic reticulum (ER) stress. Furthermore, we found that targeting NAMPT signaling, either genetically or pharmacologically, can restore the formation of beige adipocytes by increasing the number of perivascular adipocyte progenitor cells. Conversely, the absence of NAMPT signaling prevents this process. Together, our findings shed light on the mechanisms regulating the age-dependent impairment of beige adipocyte formation and underscore the E2-NAMPT-controlled ER stress pathway as a key regulator of this process.

Nuclear translocation of mitochondrial dehydrogenases as an adaptive cardioprotective mechanism.

Chemotherapy-induced cardiac damage remains a leading cause of death amongst cancer survivors. Anthracycline-induced cardiotoxicity is mediated by severe mitochondrial injury, but little is known about the mechanisms by which cardiomyocytes adaptively respond to the injury. We observed the translocation of selected mitochondrial tricarboxylic acid (TCA) cycle dehydrogenases to the nucleus as an adaptive stress response to anthracycline-cardiotoxicity in human induced pluripotent stem cell-derived cardiomyocytes and in vivo. The expression of nuclear-targeted mitochondrial dehydrogenases shifts the nuclear metabolic milieu to maintain their function both in vitro and in vivo. This protective effect is mediated by two parallel pathways: metabolite-induced chromatin accessibility and AMP-kinase (AMPK) signaling. The extent of chemotherapy-induced cardiac damage thus reflects a balance between mitochondrial injury and the protective response initiated by the nuclear pool of mitochondrial dehydrogenases. Our study identifies nuclear translocation of mitochondrial dehydrogenases as an endogenous adaptive mechanism that can be leveraged to attenuate cardiomyocyte injury.

Modulation of lncRNA links endothelial glycocalyx to vascular dysfunction of tyrosine kinase inhibitor.

AIMS: Novel cancer therapies leading to increased survivorship of cancer patients have been negated by a concomitant rise in cancer therapies-related cardiovascular toxicities. Sunitinib, a first line multi-receptor tyrosine kinase inhibitor, has been reported to cause vascular dysfunction although the initiating mechanisms contributing to this side effect remain unknown. Long non-coding RNAs (lncRNAs) are emerging regulators of biological processes in endothelial cells (ECs); however, their roles in cancer therapies-related vascular toxicities remain underexplored. METHODS AND RESULTS: We performed lncRNA expression profiling to identify potential lncRNAs that are dysregulated in human-induced pluripotent stem cell-derived ECs (iPSC-ECs) treated with sunitinib. We show that the lncRNA hyaluronan synthase 2 antisense 1 (HAS2-AS1) is significantly diminished in sunitinib-treated iPSC-ECs. Sunitinib was found to down-regulate HAS2-AS1 by an epigenetic mechanism involving hypermethylation. Depletion of HAS2-AS1 recapitulated sunitinib-induced detrimental effects on iPSC-ECs, whereas CRISPR-mediated activation of HAS2-AS1 reversed sunitinib-induced dysfunction. We confirmed that HAS2-AS1 stabilizes the expression of its sense gene HAS2 via an RNA/mRNA heteroduplex formation. Knockdown of HAS2-AS1 led to reduced synthesis of hyaluronic acid (HA) and up-regulation of ADAMTS5, an enzyme involved in extracellular matrix degradation, resulting in disruption of the endothelial glycocalyx which is critical for ECs. In vivo, sunitinib-treated mice showed reduced coronary flow reserve, accompanied by a reduction in Has2os and degradation of the endothelial glycocalyx. Finally, we identified that treatment with high molecular-weight HA can prevent the deleterious effects of sunitinib both in vitro and in vivo by preserving the endothelial glycocalyx. CONCLUSIONS: Our findings highlight the importance of lncRNA-mediated regulation of the endothelial glycocalyx as an important determinant of sunitinib-induced vascular toxicity and reveal potential novel therapeutic avenues to attenuate sunitinib-induced vascular dysfunction.

Estrogen prevents age-dependent beige adipogenesis failure through NAMPT-controlled ER stress pathway.

Thermogenic beige adipocytes are recognized as potential therapeutic targets for combating metabolic diseases. However, the metabolic advantages they offer are compromised with aging. Here, we show that treating mice with estrogen (E2), a hormone that decreases with age, to mice can counteract the aging- related decline in beige adipocyte formation when subjected to cold, while concurrently enhancing energy expenditure and improving glucose tolerance. Mechanistically, we find that nicotinamide phosphoribosyltranferase (NAMPT) plays a pivotal role in facilitating the formation of E2-induced beige adipocytes, which subsequently suppresses the onset of age-related ER stress. Furthermore, we found that targeting NAMPT signaling, either genetically or pharmacologically, can restore the formation of beige adipocytes by increasing the number of perivascular adipocyte progenitor cells. Conversely, the absence of NAMPT signaling prevents this process. In conclusion, our findings shed light on the mechanisms governing the age-dependent impairment of beige adipocyte formation and underscore the E2-NAMPT controlled ER stress as a key regulator of this process. Highlights: Estrogen restores beige adipocyte failure along with improved energy metabolism in old mice.Estrogen enhances the thermogenic gene program by mitigating age-induced ER stress.Estrogen enhances the beige adipogenesis derived from SMA+ APCs.Inhibiting the NAMPT signaling pathway abolishes estrogen-promoted beige adipogenesis.

Genetically prolonged beige fat in male mice confers long-lasting metabolic health.

A potential therapeutic target to curb obesity and diabetes is thermogenic beige adipocytes. However, beige adipocytes quickly transition into white adipocytes upon removing stimuli. Here, we define the critical role of cyclin dependent kinase inhibitor 2A (Cdkn2a) as a molecular pedal for the beige-to-white transition. Beige adipocytes lacking Cdkn2a exhibit prolonged lifespan, and male mice confer long-term metabolic protection from diet-induced obesity, along with enhanced energy expenditure and improved glucose tolerance. Mechanistically, Cdkn2a promotes the expression and activity of beclin 1 (BECN1) by directly binding to its mRNA and its negative regulator BCL2 like 1 (BCL2L1), activating autophagy and accelerating the beige-to-white transition. Reactivating autophagy by pharmacological or genetic methods abolishes beige adipocyte maintenance induced by Cdkn2a ablation. Furthermore, hyperactive BECN1 alone accelerates the beige-to-white transition in mice and human. Notably, both Cdkn2a and Becn1 exhibit striking positive correlations with adiposity. Hence, blocking Cdkn2a-mediated BECN1 activity holds therapeutic potential to sustain beige adipocytes in treating obesity and related metabolic diseases.

Alteration of the N6-methyladenosine epitranscriptomic profile in synthetic phthalate-treated human induced pluripotent stem cell-derived endothelial cells.

Background: This study aimed to characterize the N6-methyladenosine epitranscriptomic profile induced by mono(2-ethylhexyl) phthalate (MEHP) exposure using a human-induced pluripotent stem cell-derived endothelial cell model. Methods: A multiomic approach was employed by performing RNA sequencing in parallel with an N6-methyladenosine-specific microarray to identify mRNAs, lncRNAs, and miRNAs affected by MEHP exposure. Results: An integrative multiomic analysis identified relevant biological features affected by MEHP, while functional assays provided a phenotypic characterization of these effects. Transcripts regulated by the epitranscriptome were validated with quantitative PCR and methylated RNA immunoprecipitation. Conclusion: The authors' profiling of the epitranscriptome expands the scope of toxicological insights into known environmental toxins to under surveyed cellular contexts and emerging domains of regulation and is, therefore, a valuable resource to human health.

Synthetic phthalates, such as mono(2-ethyhexyl) phthalate, have long been recognized as environmental toxins. What effect these compounds have on endothelial cells remains poorly understood. To address this, the authors utilized a human-induced pluripotent stem cell-derived endothelial cell model to screen for an environmental toxin. They then obtained a profile of the epitranscriptomic changes involving the N⁶-methyladensosine modification and performed biochemical and functional assays. Overall, this study demonstrated how stem cell-based approaches can be used for toxicological screening and provided a valuable resource that profiles the epitranscriptomic response, which was complemented with RNA sequencing and functional and biochemical assays. This study provides relevant toxicological insights into the context of human health.

METTL14 Regulates Osteogenesis of Bone Marrow Mesenchymal Stem Cells via Inducing Autophagy Through m6A/IGF2BPs/Beclin-1 Signal Axis.

The development of osteoporosis is often accompanied by autophagy disturbance, which also causes new osteoblast defects from bone marrow mesenchymal stem cells (BMSCs). However, the underlying molecular mechanisms are still not fully understood. Methyltransferase-like 14 (METTL14) is the main enzyme for N6-methyladenosine (m6A), the most prevalent internal modification in mammalian mRNAs, and it has been implicated in many bioprocesses. Herein, we demonstrate that METTL14 plays a critical role in autophagy induction and hinders osteoporosis process whose expression is decreased both in human osteoporosis bone tissue and ovariectomy (OVX) mice. In vivo, METTL14+/- knockdown mice exhibit elevated bone loss and impaired autophagy similar to the OVX mice, while overexpression of METTL14 significantly promotes bone formation and inhibits the progression of osteoporosis caused by OVX surgery. In vitro, METTL14 overexpression significantly enhances the osteogenic differentiation ability of BMSCs through regulating the expression of beclin-1 depending on m6A modification and inducing autophagy; the opposite is true with METTL14 silencing. Subsequently, m6A-binding proteins IGF2BP1/2/3 recognize m6A-methylated beclin-1 mRNA and promote its translation via mediating RNA stabilization. Furthermore, METTL14 negatively regulates osteoclast differentiation. Collectively, our study reveals the METTL14/IGF2BPs/beclin-1 signal axis in BMSCs osteogenic differentiation and highlights the critical roles of METTL14-mediated m6A modification in osteoporosis.

METTL3 induces bone marrow mesenchymal stem cells osteogenic differentiation and migration through facilitating M1 macrophage differentiation.

Despite the crucial role of m6A methyltransferase METTL3 in multiple diseases onset and progression, there are still lacking hard evidence proving that METTL3 could affect macrophage polarization in the stage of bone repair. Here, we aimed to explore the potential involvement of METTL3 in bone repair through modulating macrophage polarization and decipher the underlying cellular/molecular mechanisms. Here we treated RAW 264.7 cells and BM-derived primary macrophages (BMDM) with lipopolysaccharide (LPS) to induce M1 differentiation. METTL3 expression was upregulated in pro-inflammatory macrophages (M1) as compared with macrophages (M0). And overexpression of METTL3 promoted the expression of IL-6 and iNOS secretion by M1 macrophage. In the coculture condition, M1 macrophages with forced expression of METTL3 significantly enhanced migration ability of BMSCs, and also remarkably facilitated osteogenesis ability of BMSCs; the opposite was true when expression of METTL3 was knockdown. In addition, the m6A-RIP microarray suggested that METTL3 silencing significantly reduce the m6A modification of DUSP14, HDAC5 and Nfam1. Furthermore, the findings showed that expression of HADC5 was downregulated in M1 macrophages with METTL3 knockdown, while the DUSP14 expression had slight change and Nfam1 expression was very low. In contrast, METTL3 overexpression promoted HDAC5 expression, indicating that HDAC5 is the critical target gene of METTL3. Under such a theme, we proposed that METTL3 overexpression might be a new approach of replacement therapy for the treatment of bone repair.

ALKBH5 suppresses tumor progression via an m6A-dependent epigenetic silencing of pre-miR-181b-1/YAP signaling axis in osteosarcoma.

ALKBH5 is the main enzyme for m6A-based demethylation of RNAs and it has been implicated in many biological and pathophysiological processes. Here, we aimed to explore the potential involvement of ALKBH5 in osteosarcoma and decipher the underlying cellular/molecular mechanisms. We discovered downregulated levels of demethylase ALKBH5 were correlated with increased m6A methylation in osteosarcoma cells/tissues compared with normal osteoblasts cells/tissues. ALKBH5 overexpression significantly suppressed osteosarcoma cell growth, migration, invasion, and trigged cell apoptosis. In contrast, inhibition of ALKBH5 produced the opposite effects. Whereas ALKBH5 silence enhanced m6A methylations of pre-miR-181b-1 and YAP-mRNA exerting oncogenic functions in osteosarcoma. Moreover, upregulation of YAP or downregulation of mature miR-181b-5p displayed a remarkable attenuation of anti-tumor activities caused by ALKBH5. Further results revealed that m6A methylated pre-miR-181b-1 was subsequently recognized by m6A-binding protein YTHDF2 to mediate RNA degradation. However, methylated YAP transcripts were recognized by YTHDF1 to promote its translation. Therefore, ALKBH5-based m6A demethylation suppressed osteosarcoma cancer progression through m6A-based direct/indirect regulation of YAP. Thus, ALKBH5 overexpression might be considered a new approach of replacement therapy for osteosarcoma treatment.

ALKBH5-mediated m6A mRNA methylation governs human embryonic stem cell cardiac commitment.

N6-methyladenosine (m6A), as the most abundant modification of mammalian messenger RNAs, is essential for tissue development and pathogenesis. However, the biological significance of m6A methylation in cardiac differentiation and development remains largely unknown. Here, we identify that the downregulation of m6A demethylase ALKBH5 is responsible for the increase of m6A methylation and cardiomyocyte fate determination of human embryonic stem cells (hESCs) from mesoderm cells (MESs). In contrast, ALKBH5 overexpression remarkably blocks cardiomyocyte differentiation of hESCs. Mechanistically, KDM5B and RBBP5, the components of H3K4 modifying enzyme complexes, are identified as downstream targets for ALKBH5 in cardiac-committed hESCs. Loss of function of ALKBH5 alters the expression of KDM5B and RBBP5 through impairing stability of their mRNAs, which in turn promotes the transcription of GATA4 by enhancing histone H3 Lys4 trimethylation (H3K4me3) at the promoter region of GATA4. Taken together, we reveal a previously unidentified role of m6A demethylase ALKBH5 in determining cardiac lineage commitment of hESCs.