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The m6A methyltransferase METTL16 negatively regulates MCP1 expression in mesenchymal stem cells during monocyte recruitment.

ABSTRACT: Mesenchymal stem cells (MSCs) possess strong immunoregulatory functions, one aspect of which is recruiting monocytes from peripheral vessels to local tissue by secreting monocyte chemoattractant protein 1 (MCP1). However, the regulatory mechanisms of MCP1 secretion in MSCs are still unclear. Recently, the N6-methyladenosine (m6A) modification was reported to be involved in the functional regulation of MSCs. In this study, we demonstrated that methyltransferase-like 16 (METTL16) negatively regulated MCP1 expression in MSCs through the m6A modification. Specifically, the expression of METTL16 in MSCs decreased gradually and was negatively correlated with the expression of MCP1 after coculture with monocytes. Knocking down METTL16 markedly enhanced MCP1 expression and the ability to recruit monocytes. Mechanistically, knocking down METTL16 decreased MCP1 mRNA degradation, which was mediated by the m6A reader YTH N6-methyladenosine RNA-binding protein 2 (YTHDF2). We further revealed that YTHDF2 specifically recognized m6A sites on MCP1 mRNA in the CDS region and thus negatively regulated MCP1 expression. Moreover, an in vivo assay showed that MSCs transfected with METTL16 siRNA showed greater ability to recruit monocytes. These findings reveal a potential mechanism by which the m6A methylase METTL16 regulates MCP1 expression through YTHDF2-mediated mRNA degradation and suggest a potential strategy to manipulate MCP1 expression in MSCs.

SUBMITTER: Zhang Z

PROVIDER: S-EPMC10070103 | biostudies-literature | 2023 Mar

REPOSITORIES: biostudies-literature

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The m6A methyltransferase METTL16 negatively regulates MCP1 expression in mesenchymal stem cells during monocyte recruitment.

Zhang Zhaoqiang Z Xie Zhongyu Z Lin Jiajie J Sun Zehang Z Li Zhikun Z Yu Wenhui W Zeng Yipeng Y Ye Guiwen G Li Jinteng J Ye Feng F Su Zepeng Z Che Yunshu Y Xu Peitao P Zeng Chenying C Wang Peng P Wu Yanfeng Y Shen Huiyong H

JCI insight 20230322 6

Mesenchymal stem cells (MSCs) possess strong immunoregulatory functions, one aspect of which is recruiting monocytes from peripheral vessels to local tissue by secreting monocyte chemoattractant protein 1 (MCP1). However, the regulatory mechanisms of MCP1 secretion in MSCs are still unclear. Recently, the N6-methyladenosine (m6A) modification was reported to be involved in the functional regulation of MSCs. In this study, we demonstrated that methyltransferase-like 16 (METTL16) negatively regula ...[more]

PMID: 36795489

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Project description:BackgroundMesenchymal stem cells (MSCs) exhibit two bidirectional immunomodulatory abilities: proinflammatory and anti-inflammatory regulatory effects. Long noncoding RNAs (lncRNAs) have important functions in the immune system. Previously, we performed high-throughput sequencing comparing lncRNA expression profiles between MSCs cocultured with or without CD14+ monocytes and screened out a new lncRNA termed lncRNA MCP1 regulatory factor (MRF). However, the mechanism of MRF in MSCs is still unknown.MethodsMRF expression was quantified via qRT-PCR. RNA interference and lentiviruses were used to regulate MRF expression. The immunomodulatory effects of MSCs on monocytes were evaluated via monocyte migration and macrophage polarization assays. RNA pull-down and mass spectrometry were utilized to identify downstream factors of MRF. A dual-luciferase reporter assay was applied to analyze the transcription factors regulating MRF. qRT-PCR, western blotting and ELISAs were used to assess MCP1 expression. A human monocyte adoptive transfer mouse model was applied to verify the function of MRF in vivo.ResultsMRF was upregulated in MSCs during coculture with CD14+ monocytes. MRF increased monocyte recruitment by upregulating the expression of monocyte chemotactic protein (MCP1). Knockdown of MRF enhanced the regulatory effect of MSCs on restraining M1 polarization and facilitating M2 polarization. Mechanistically, MRF bound to the downstream protein heterogeneous nuclear ribonucleoprotein D (HNRNPD) to upregulate MCP1 expression, and the transcription factor interferon regulatory factor 1 (IRF1) activated MRF transcription early during coculture. The human monocyte adoptive transfer model showed that MRF downregulation in MSCs inhibited monocyte chemotaxis and enhanced the effects of MSCs to inhibit M1 macrophage polarization and promote M2 polarization in vivo.ConclusionWe identified the new lncRNA MRF, which exhibits proinflammatory characteristics. MRF regulates the ability of MSCs to accelerate monocyte recruitment and modulate macrophage polarization through the HNRNPD-MCP1 axis and initiates the proinflammatory regulatory process in MSCs, suggesting that MRF is a potential target to improve the clinical effect of MSC-based therapy or correct MSC-related immunomodulatory dysfunction under pathological conditions.

Project description:Haemoglobin H (HbH) disease is caused by a disorder of α-globin synthesis, and it results in a wide range of clinical symptoms. M6A methylation modification may be one of the mechanisms of heterogeneity. Therefore, this article explored the role of methyltransferase like 16 (METTL16) in HbH disease. The results of epigenetic transcriptome microarray were analysed and verified through bioinformatic methods and qRT-PCR, respectively. The overexpression or knock down of METTL16 in K562 cells was examined to determine its role in reactive oxygen species (ROS), cell cycle processes or iron overload. YTH domain family protein 3 (YTHDF3) was knocked down in K562 cells and K562 cells overexpressing METTL16 via siRNA to investigate its function. In addition, haemoglobin expression was detected through benzidine staining. qRT-PCR, WB, methylated RNA Immunoprecipitation (MeRIP) and (RNA Immunoprecipitation) RIP experiments were conducted to explore the mechanism of intermolecular interaction. METTL16, YTHDF3 and solute carrier family 5 member 3 (SLC5A3) mRNA and the methylation level of SLC5A3 mRNA were downregulated in HbH patients. Insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) mRNA expression was negatively correlated with HGB content among patients with HbH-CS disease. Overexpression of METTL16 increased ROS and intracellular iron contents in K562 cells, changed the K562 cell cycle, reduced hemin-induced haemoglobin synthesis, increased the expressions of SLC5A3 and HBG and increased SLC5A3 mRNA methylation levels. Knockdown of METTL16 reduced ROS and intracellular iron contents in K562 cells. Hemin treatment of K562 cells for more than 14 days reduced the protein expressions of METTL16 and SLC5A3 and SLC5A3 mRNA methylation levels. Knockdown of YTHDF3 rescued the intracellular iron content changes induced by the overexpression of METTL16. The RIP experiment revealed that SLC5A3 mRNA can be enriched by METTL16 antibody. METTL16 may affect the expression of SLC5A3 by changing its m6A modification level and regulating ROS synthesis, intracellular iron and cycle of red blood cells. Moreover, METTL16 possibly affects the expression of haemoglobin through IGF2BP3, which regulates the clinical phenotype of HbH disease.

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The m6A methyltransferase METTL16 negatively regulates MCP1 expression in mesenchymal stem cells during monocyte recruitment.

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The m6A methyltransferase METTL16 negatively regulates MCP1 expression in mesenchymal stem cells during monocyte recruitment.

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