FTO regulates adipogenesis by controlling cell cycle progression via m6A-YTHDF2 dependent mechanism

N⁶-methyladenosine (m⁶A) is the most prevalent internal mRNA modification in eukaryotes. Loss of m⁶A demethylase FTO increases m⁶A levels and inhibits adipogenesis of preadipocytes. However, its underlying mechanism remains elusive. Here, we demonstrated that silencing FTO inhibited adipogenesis of preadipocytes through impairing cell cycle progression at the early stage of adipogenesis. FTO knockdown markedly decreased the expression of CCNA2 and CDK2, crucial cell cycle regulators, leading to delayed entry of MDI-induced cells into G2 phase. Furthermore, the m⁶A levels of CCNA2 and CDK2 mRNA were significantly upregulated following FTO knockdown. m⁶A-binding protein YTHDF2 recognized and decayed methylated mRNAs of CCNA2 and CDK2, leading to decreased protein expression, thereby prolonging cell cycle progression and suppressing adipogenesis. Our work unravels that FTO regulates adipogenesis by controlling cell cycle progression in an m⁶A-YTHDF2 dependent manner, which provides insights into critical roles of m⁶A methylation in adipogenesis.     

YTHDF2 Suppresses Notch Signaling through Post-transcriptional Regulation on Notch1

YTH domain family 2 (YTHDF2) is an N6-methyladenosine (m⁶A) binding protein promoting mRNA degradation in various biological processes. Despite its essential roles, the role of YTHDF2 in determining cell fates has not been fully elucidated. Notch signaling plays a vital role in determining cell fates, such as proliferation, differentiation, and apoptosis. We investigated the effect of YTHDF2 on Notch signaling. Our results show that YTHDF2 inhibits Notch signaling by downregulating the Notch1, HES1, and HES5 mRNA levels. Analyzing YTHDF2 deletion mutants indicates that the YTH domain is critical in regulating the Notch signal by directly binding m⁶A of Notch1 mRNA. Recently, YTHDF2 nuclear translocation was reported under heat shock conditions, but its physiological function is unknown. In our study, the YTH domain is required for YTHDF2 nuclear translocation. In addition, under heat shock stress, the Notch signal was significantly restored due to the increased expression of the Notch1 targets. These results suggest that YTHDF2 in the cytoplasm may act as an intrinsic suppressor in Notch signaling by promoting Notch1 mRNA degradation under normal cellular conditions. Conversely, upon the extracellular stress such as heat shock, YTHDF2 nuclear translocation resulting in reduced Notch1 mRNA decay may contribute to the increasing of Notch intracellular domain (NICD) regulating the survival-related target genes.     

SIRT6 overexpression inhibits cementogenesis by suppressing glucose transporter 1

Cementum, which shares common features with bone in terms of biochemical composition, is important for the homeostasis of periodontium during periodontitis and orthodontic treatment. Sirtuin 6 (SIRT6), as a member of the sirtuin family, plays key roles in the osteogenic differentiation of bone marrow mesenchymal stem cells. However, the involvement of SIRT6 in cementoblast differentiation and mineralization and the underlying mechanisms remain unknown. In this study, we observed that the expression of SIRT6 increased during cementoblast differentiation initially. Analysis of the gain- and loss-of-function indicated that overexpressing SIRT6 in OCCM-30 cells suppresses cementoblast differentiation and mineralization and downregulating SIRT6 promotes cementogenesis. GLUT1, a glucose transporter necessary in cementogenesis, was inhibited by SIRT6. Overexpressing GLUT1 in SIRT6-overexpressed OCCM-30 cells rescued the inhibitory effect of SIRT6 on cementoblast differentiation and mineralization. Moreover, AMPK was activated after overexpressing SIRT6 and inhibited cementoblast differentiation and mineralization. Downregulating the expression of SIRT6 inhibited AMPK activity. Meanwhile, GLUT1 overexpression significantly decreased AMPK activity. Overall, on one hand, SIRT6 inhibited cementoblast differentiation and mineralization by suppressing GLUT1. On the other hand, SIRT6 inhibited cementoblast differentiation and mineralization by activating the AMPK pathway. GLUT1 overexpression also rescued the increased AMPK pathway activated by SIRT6.     

YTHDF2 mediates LPS-induced osteoclastogenesis and inflammatory response via the NF-κB and MAPK signaling pathways

Aberrant elevation of osteoclast differentiation and function is responsible for disrupting bone homeostasis in various inflammatory bone diseases. YTH domain family 2 (YTHDF2) is a well-known m⁶A-binding protein that plays an essential role in regulating cell differentiation and inflammatory processes by mediating mRNA degradation. However, the regulatory role of YTHDF2 in inflammatory osteoclast differentiation remains unelucidated. Here, we detected the expression of m⁶A-related genes and found that YTHDF2 was upregulated in RANKL-primed osteoclast precursors stimulated with lipopolysaccharide (LPS). Ythdf2 knockdown in RAW264.7 cells and primary bone marrow-derived macrophages (BMMs) enhanced osteoclast formation and bone resorption, which was assessed by TRAP staining assay and pit formation assay. Ythdf2 depletion upregulated osteoclast-related gene expression and proinflammatory cytokine secretion. In contrast, overexpression of Ythdf2 produced the reverse effect. Furthermore, Ythdf2 knockdown enhanced the phosphorylation of IKKα/β, IκBα, ERK, P38 and JNK. NF-κB and MAPK signaling pathway inhibitors effectively abrogated the enhanced expression of Nfact1, c-Fos, IL-1β and TNF-α caused by Ythdf2 knockdown. Mechanistically, the mRNA stability assay revealed that Ythdf2 depletion led to stabilization of Tnfrsf11a, Traf6, Map4k4, Map2k3, Map2k4 and Nfatc1 mRNA. In summary, our findings demonstrated that YTHDF2 has a negative regulatory role in LPS-induced osteoclast differentiation and the inflammatory response via the NF-κB and MAPK signaling pathways.     

N6-methyladenosine demethylase FTO promotes M1 and M2 macrophage activation

Macrophage polarization is the driving force of various inflammatory diseases, especially those involved in M1/M2 imbalance. N6-methyladenosine (m⁶A) is the most prevalent internal mRNA modification in eukaryotes that affects multiple biological processes, including those involved developmental arrest and immune response. However, the role of m⁶A in macrophage polarization remains unclear. This study found that FTO silencing significantly suppressed both M1 and M2 polarization. FTO depletion decreased the phosphorylation levels of IKKα/β, IκBα and p65 in the NF-κB signaling pathway. The expression of STAT1 was downregulated in M1-polarized macrophages while the expression of STAT6 and PPAR-γ decreased in M2 polarization after FTO knockdown. The actinomycin D experiments showed that FTO knockdown accelerated mRNA decay of STAT1 and PPAR-γ. Furthermore, the stability and expression of STAT1 and PPAR-γ mRNAs increased when the m⁶A reader YTHDF2 was silenced. In conclusion, our results suggest that FTO knockdown inhibits the NF-κB signaling pathway and reduces the mRNA stability of STAT1 and PPAR-γ via YTHDF2 involvement, thereby impeding macrophage activation. These findings indicated a previously unrecognized link between FTO and macrophage polarization and might open new avenues for research into the molecular mechanisms of macrophage polarization-related diseases.     

Meclofenamic acid selectively inhibits FTO demethylation of m6A over ALKBH5

Two human demethylases, the fat mass and obesity-associated (FTO) enzyme and ALKBH5, oxidatively demethylate abundant N⁶-methyladenosine (m⁶A) residues in mRNA. Achieving a method for selective inhibition of FTO over ALKBH5 remains a challenge, however. Here, we have identified meclofenamic acid (MA) as a highly selective inhibitor of FTO. MA is a non-steroidal, anti-inflammatory drug that mechanistic studies indicate competes with FTO binding for the m⁶A-containing nucleic acid. The structure of FTO/MA has revealed much about the inhibitory function of FTO. Our newfound understanding, revealed herein, of the part of the nucleotide recognition lid (NRL) in FTO, for example, has helped elucidate the principles behind the selectivity of FTO over ALKBH5. Treatment of HeLa cells with the ethyl ester form of MA (MA2) has led to elevated levels of m⁶A modification in mRNA. Our collective results highlight the development of functional probes of the FTO enzyme that will (i) enable future biological studies and (ii) pave the way for the rational design of potent and specific inhibitors of FTO for use in medicine.     

The role of RNA adenosine demethylases in the control of gene expression

RNA modifications are being recognized as an essential factor in gene expression regulation. They play essential roles in germ line development, differentiation and disease. In eukaryotic mRNAs, N⁶-adenosine methylation (m⁶A) is the most prevalent internal chemical modification identified to date. The m⁶A pathway involves factors called writers, readers and erasers. m⁶A thus offers an interesting concept of dynamic reversible modification with implications in fine-tuning the cellular metabolism. In mammals, FTO and ALKBH5 have been initially identified as m⁶A erasers. Recently, FTO m⁶A specificity has been debated as new reports identify FTO targeting N⁶,2′-O-dimethyladenosine (m⁶A_m). The two adenosine demethylases have diverse roles in the metabolism of mRNAs and their activity is involved in key processes, such as embryogenesis, disease or infection. In this article, we review the current knowledge of their function and mechanisms and discuss the existing contradictions in the field. This article is part of a Special Issue entitled: mRNA modifications in gene expression control edited by Dr. Soller Matthias and Dr. Fray Rupert.     

Fat mass and obesity-associated protein regulates lipogenesis via m6A modification in fatty acid synthase mRNA

As the first identified N⁶-methyladenosine (m⁶A) demethylase, fat mass and obesity-associated (FTO) protein is associated with fatty acid synthase (FASN) and lipid accumulation. However, little is known about the regulatory role of FTO in the expression of FASN and de novo lipogenesis through m⁶A modification. In this study, we used FTO small interfering RNA to explore the effects of FTO knockdown on hepatic lipogenesis and its underlying epigenetic mechanism in HepG2 cells. We found that knockdown of FTO increased m⁶A levels in total RNA and enhanced the expression of YTH domain family member 2 which serves as the m⁶A-binding protein. The de novo lipogenic enzymes and intracellular lipid content were significantly decreased under FTO knockdown. Mechanistically, knockdown of FTO dramatically enhanced m⁶A levels in FASN messenger RNA (mRNA), leading to the reduced expression of FASN mRNA through m⁶A-mediated mRNA decay. The protein expressions of FASN along with acetyl CoA carboxylase and ATP-citrate lyase were further decreased, which inhibited de novo lipogenesis, thereby resulting in the deficiency of lipid accumulation in HepG2 cells and the induction of cellular apoptosis. The results reveal that FTO regulates hepatic lipogenesis via FTO-dependent m⁶A demethylation in FASN mRNA and indicate the critical role of FTO-mediated lipid metabolism in the survival of HepG2 cells. This study provides novel insights into a unique RNA epigenetic mechanism by which FTO mediates hepatic lipid accumulation through m⁶A modification and indicates that FTO could be a potential target for obesity-related diseases and cancer.