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. 2018 Jul 30;38(16):e00116-18.
doi: 10.1128/MCB.00116-18. Print 2018 Aug 15.

The RNA Methyltransferase Complex of WTAP, METTL3, and METTL14 Regulates Mitotic Clonal Expansion in Adipogenesis

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Free PMC article

The RNA Methyltransferase Complex of WTAP, METTL3, and METTL14 Regulates Mitotic Clonal Expansion in Adipogenesis

Masatoshi Kobayashi et al. Mol Cell Biol. .
Free PMC article

Abstract

Adipocyte differentiation is regulated by various mechanisms, of which mitotic clonal expansion (MCE) is a key step. Although this process is known to be regulated by cell cycle modulators, the precise mechanism remains unclear. N6-Methyladenosine (m6A) posttranscriptional RNA modification, whose methylation and demethylation are performed by respective enzyme molecules, has recently been suggested to be involved in the regulation of adipogenesis. Here, we show that an RNA N6-adenosine methyltransferase complex consisting of Wilms' tumor 1-associating protein (WTAP), methyltransferase like 3 (METTL3), and METTL14 positively controls adipogenesis by promoting cell cycle transition in MCE during adipogenesis. WTAP, coupled with METTL3 and METTL14, is increased and distributed in nucleus by the induction of adipogenesis dependently on RNA in vitro Knockdown of each of these three proteins leads to cell cycle arrest and impaired adipogenesis associated with suppression of cyclin A2 upregulation during MCE, whose knockdown also impairs adipogenesis. Consistent with this, Wtap heterozygous knockout mice are protected from diet-induced obesity with smaller size and number of adipocytes, leading to improved insulin sensitivity. These data provide a mechanism for adipogenesis through the WTAP-METTL3-METTL14 complex and a potential strategy for treatment of obesity and associated disorders.

Keywords: adipocyte; diabetes; obesity.

Figures

FIG 1
FIG 1
WTAP is required in adipocyte differentiation in vitro. (A to D) In 3T3-L1 cells WTAP was knocked down by infection of adeno-shWTAP and induced to differentiate to adipocytes (indicated as time zero). (A) mRNA expression levels of Wtap during 10 days after DMI (n = 4). (B) The protein levels of WTAP and cyclin A2 were analyzed by Western blotting and quantified with densitometry. C, adeno-control; W, adeno-shWTAP. (C) Pparg and its related gene expression at day 2 (n = 4). (D) Oil Red O staining with the indicated doses of adeno-shWTAP or control adenovirus at day 10. MOI, multiplicity of infection. (E to G) Embryonic fibroblasts from Wtap+/− (MEF-Wtap+/−) and WT (MEF-WT) mice were induced to differentiate into adipocytes with DMI. (E) mRNA expression levels of Wtap before and after DMI (indicated as time zero) (n = 4). (F) The protein levels of WTAP were analyzed by Western blotting and quantified with densitometry. (G) Pparg and its related gene expression at day 2 (n = 4). Data represent means ± SEM (A and E) or means + SEM (C and G). *, P < 0.05; **, P < 0.01; ***, P < 0.001.
FIG 2
FIG 2
Cyclin A2 is upregulated during MCE of adipocyte differentiation in vitro and in the development of obesity in vivo. (A to D) The mRNA expressions of cyclin A2 (Ccna2) (A and C) and Wtap (B and D) in 3T3-L1 (A and B) and F442A (C and D) cells during adipocyte differentiation. Day 0 indicates the induction by standard DMI (n = 3). (E to H) The mRNA expressions of Ccna2 (E and G) and Wtap (F and H) in the WAT of DIO mice (E and F) and db/db mice (G and H). C57BL/6 mice fed on HFD for the duration of the indicated weeks were compared to control mice fed on NCD (E and F), and db/db mice were compared to control m/m mice at the indicated ages (G and H) (n = 5). Data represent means ± SEM (A to D) or means + SEM (E to H). **, P < 0.01.
FIG 3
FIG 3
Cyclin A2 is required for cell cycle transition of MCE in adipocyte differentiation in vitro. Cyclin A2 was knocked down in 3T3-L1 cells with siRNA and induced to differentiate into adipocytes. (A and B) Cyclin A2 was reduced especially during the MCE period (day 0 to 2), shown at the mRNA (n = 3) (A) and protein (B) levels (at day 1). (C) Cell cycle analysis with flow cytometry at 40 h after standard DMI induction (n = 6). The typical data are shown in the upper graphs, and the lower graphs show the percentage of the distribution in each cell cycle period. (D and E) Adipocyte differentiation was evaluated by mRNA expression of Cebpa and Pparg at day 4 (n = 3) (D) and Oil Red O staining at day 10 (E). Data represent means ± SEM (A) or means + SEM (C and D). *, P < 0.05; **, P < 0.01.
FIG 4
FIG 4
Knockdown of WTAP impairs cyclin A2 upregulation and arrests cell cycle transition during MCE of adipocyte differentiation in vitro. (A and B) Cyclin A2 mRNA (A) and protein (B) levels in the knockdown of WTAP with adeno-shWTAP in the same experiment as that shown in Fig. 1A and B, respectively. The protein levels of cyclin A2 were analyzed by Western blotting and quantified with densitometry. C, adeno-control; W, adeno-shWTAP. (C) Flow cytometric analysis of the cell cycle of adeno-shWTAP or control adenovirus-infected 3T3-L1 cells 40 h after DMI treatment. The typical data are shown in the upper graphs, and the lower graphs show the percentage of the distribution of each cell cycle period (n = 6). The left and right graphs indicate the cells without and with the induction of differentiation by DMI, respectively. (D and E) Cyclin A2 mRNA (D) and protein (E) levels in MEF-Wtap+/− and MEF-WT mice in the same experiment as that shown in Fig. 1E and F, respectively. The protein levels of cyclin A2 were analyzed by Western blotting and quantified with densitometry. (F) Flow cytometric analysis of the cell cycle of MEF-Wtap+/− and MEF-WT 40 h after DMI. The typical data are shown in the upper graphs, and the lower graphs show the percentage of the distribution in each cell cycle period (n = 4 to 6). (A, B, D, and E) Cyclin A2 mRNA and protein levels were shown as a ratio to the baseline. Data represent means ± SEM (A and D) or means + SEM (C and F). *, P < 0.05; **, P < 0.01; ***, P < 0.001.
FIG 5
FIG 5
WTAP physically interacts with METTL3 and METTL14. (A and B) GST pulldown assay. (A) Schematic constructs of GST-fused full-length WTAP and its deletion mutant proteins as bait proteins. The numbers represent the amino acid numbers for the sequences of the proteins. (B) FLAG-METTL3- and FLAG-METTL14-overexpressed COS cell lysates, as prey proteins, were incubated with the immobilized bait proteins. The eluted protein complex was immunoblotted with anti-METTL3, anti-METTL14, and anti-FLAG M2 antibody. (C) The molecular structure of human and mouse WTAP based on Ensembl. The boxed sequence represents the glutamine-rich region, which corresponds to the middle region shown in Fig. 6A, with shaded glutamine residues (Q). Four coiled-coil domains are shown with underlining. A total of 96% amid acid sequence is conserved between mouse and human, with 100% conservation in the middle and N-terminal regions.
FIG 6
FIG 6
Immunofluorescence staining of WTAP, METTL3, and METTL14 in nuclei is enhanced in adipocyte differentiation of 3T3-L1 cells. Immunofluorescence study in 3T3-L1 cells, shown with METTL3-Alexa Fluor 488 (AF488) (green), METTL14-AF594 (red), WTAP-AF647 (blue), and DNA-DAPI (cyan). (A and B) The time course of METTL3, METTL14, and WTAP staining during adipocyte differentiation by DMI induction. (C) The merged images of DAPI with METTL3, METTL14, and WTAP in the cell without DMI induction. Scale bars, 50 μm (A) and 10 μm (B and C).
FIG 7
FIG 7
WTAP, METTL3, and METTL14 are increased and distributed in nucleus by DMI stimulation, similar to other nuclear speckle proteins, in vitro. (A) The mRNA expression of Mettl3, Mettl14, and Wtap for 48 h after DMI stimulation. Data represent means ± SEM. P < 0.05 (*) and P < 0.01 (**) for DMI (−) versus DMI (+) at each time point. (B and C) The cytoplasmic and nuclear protein levels of METTL3, METTL14, and WTAP were analyzed by Western blotting (B) and quantified by densitometry (C). (D and E) Immunofluorescence study for nuclear speckle marker protein SC-35 (D) and Smith antigen (E) in 3T3-L1 cells 24 h after DMI stimulation. Scale bars, 50 μm (left) and 10 μm (right).
FIG 8
FIG 8
WTAP may recruit METTL3 and METTL14 to RNA in adipocyte differentiation in vitro. (A) 3T3-L1 cells 48 h after the induction by DMI were harvested and treated with RNase or left untreated. Their cytosolic, nuclear, and total proteins were extracted and immunoblotted with anti-WTAP antibody. Note that the increase of nuclear WTAP by DMI was canceled by RNase treatment. The fractionation of nuclear and cytosolic protein was validated by immunoblotting of lamin A/C and α-tubulin, respectively. (B and C) The effect of knockdown of METTL3, METTL14, and WTAP 24 h after DMI treatment in 3T3-L1 cells. (B) The nuclear protein levels analyzed by Western blotting. (C) The immunofluorescence study, shown with METTL3-Alexa Fluor 488 (AF488) (green), METTL14-AF594 (red), WTAP-AF647 (blue), and DNA-DAPI (cyan). Scale bars, 50 μm (left) and 10 μm (right).
FIG 9
FIG 9
WMM complex regulates the cell cycle transition during MCE. (A to G) Single or double knockdown of METTL3 and/or METTL14 by siRNA in 3T3-L1 cells. (A and B) Mettl3 (A) and Mettl14 (B) mRNA expression levels after the stimulation of adipocyte differentiation with DMI (n = 4). (C) The protein levels of METTL3, METTL14, and WTAP 24 h after DMI treatment. (D to G) The effects of METTL3 and/or METTL14 knockdown on differentiation into adipocytes. (D) Ccna2 mRNA expression after DMI, shown as a ratio to the baseline (n = 4). (E) Flow cytometric analysis of the cell cycle 40 h after DMI treatment (n = 5). (F) mRNA expression of Pparg and its related genes 48 h after DMI (n = 4). (G) Oil Red O staining 10 days after DMI. (H) Cooverexpression of METTL3-FLAG and METTL14-FLAG in COS cells. The empty pEZ vector was used as the control vector. In the FLAG blotting, the upper bands (*) represent FLAG-METTL3, and the lower bands (**) represent FLAG-METTL14. Data represent means (A, B, and D) or means + SEM (E and F). (D) P < 0.05 (*) and P < 0.05 (**) for siControl versus siMETTL3. P < 0.05 (†) and P < 0.01 (††) for siControl versus siMETTL14. P < 0.05 (§) and P < 0.01 (§§) for siControl versus siMETTL3 and siMETTL14. (E and F) *, P < 0.05; **, P < 0.05.
FIG 10
FIG 10
Wtap+/− mice are resistant to HFD-induced obesity, with small size and number of adipocytes. (A and B) The time course of the change in body weight (BW) of mice fed on NCD (n = 7 to 14) (A) and HFD (n = 7 or 8) (B) (HFD during 8 to 28 weeks of age). (C) The body length of Wtap+/− and WT mice. The mice were 16 weeks old and fed on HFD for 6 weeks (n = 7 to 10). (D to M) Wtap+/− and WT mice were fed on HFD or NCD for 2 weeks (during 11 to 13 weeks of age), except for those depicted in Fig. 5K, which were fed on HFD for 6 weeks (during 11 to 17 weeks of age) and sacrificed for the following analysis. (D) Total body weight. (E and F) Lean mass (E) and fat mass (F) as measured by DEXA. (G and H) The weight of eWAT (G) and scWAT (H) (n = 10 or 11). (I to K) Averaged distribution of adipocyte size as measured by a Coulter counter in the eWAT from wild-type (blue) and Wtap+/− (red) mice. (L) Average volume of one adipocyte. (M) Average number of adipocytes in the eWAT of a mouse (n = 7 to 11). (N to P) The gene expression of Wtap (N), Ccna2 (O), and Pparg (P) in stromal vascular cells (SVCs) and the adipocyte fraction of eWAT from mice fed on HFD for 2 weeks at 11 to 13 weeks of age compared to those from mice fed on NCD (n = 8). (Q) Flow cytometric analysis of the cell cycle for the lineage and CD31 double-negative population of SVCs at the second passage (more than 96% of total SVCs) 20 h after DMI treatment. The typical data are shown in the upper graphs, and the lower graphs show the percentage of the distribution in each cell cycle period (n = 8 to 10). Data represent means ± SEM (A and B) or means + SEM (C to Q). *, P < 0.05; **, P < 0.01; ***, P < 0.001.
FIG 11
FIG 11
Increased energy expenditure and improved leptin sensitivity and circulating lipid profiles in Wtap+/ mice. (A) Food intake at 28 weeks of age on HFD (n = 5). (B) O2 consumption measured in 7- to 9-week-old mice fed on HFD (n = 10). (C and F) The expression of brown fat-related genes in BAT (C) and scWAT (F) in 13-week-old mice fed on HFD for 2 weeks (n = 7 or 8). (D, E, G, and H) UCP-1 protein levels analyzed by Western blotting (n = 6) (D and G) and immunohistochemistry (E and H) in BAT (D and E) and scWAT (G and H) of 15-week-old mice fed on HFD for 2 weeks. Scale bars, 500 μm. (I) Plasma leptin levels in 16-week-old mice fed on NCD and 28-week-old mice fed on HFD for 20 weeks (n = 6 to 8). (J) The gene expression of neuropeptides associated with appetite in the hypothalamus in 28-week-old mice fed on HFD for 20 weeks (n = 8). (K to M) Plasma lipid profile from the mice fed on HFD for 20 weeks at 28 weeks of age (n = 6 to 15). Shown are total cholesterol (T. Chol) (K), triglycerides (TG) (L), and free fatty acids (FFA) (M). Data represent means + SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
FIG 12
FIG 12
Hepatic steatosis and macrophage infiltration in both adipose tissue and liver were attenuated in Wtap+/ mice. (A) Liver weight in 28-week-old mice on HFD for 20 weeks (n = 6). (B) H&E staining of liver from 28-week-old mice on HFD for 20 weeks. A representative section of each genotype is shown. Scale bar, 100 μm. (C) TG content of liver from 28-week-old mice on HFD for 20 weeks (n = 8). (D) Plasma MCP-1 levels in 28-week-old mice on HFD for 20 weeks (n = 6 to 8). (E and F) Immunohistochemistry with anti-F4/80 antibody in eWAT (E) and the ratios of anti-F4/80 antibody-positive nucleus counts (F) in 28-week-old mice on HFD for 20 weeks. Immunoreactivity for F4/80 is visualized as brown, whereas routine hematoxylin staining appears blue. Scale bars, 100 μm (n = 4 to 8). (G to I) The expression of macrophage-related and proinflammatory genes in the eWAT from 20-week-old mice on HFD for 12 weeks. Shown are Emr1 (F4/80) and Cd68 (G), an M1-like macrophage-related gene, Itgax (coding for CD11c) (H), and Ccl2 (MCP-1), Ccr2, and Tnf (I) (n = 6 to 10). (J and K) Immunohistochemistry with anti-F4/80 antibody in liver (J) and the ratios of F4/80-positive nucleus counts (K) in 28-week-old mice on HFD for 20 weeks. Immunoreactivity for F4/80 is visualized as brown, whereas routine hematoxylin staining appears blue. Scale bars, 100 μm (n = 7 or 8). (L to N) The expression of macrophage-related and proinflammatory genes in liver from 20-week-old mice on HFD for 12 weeks. Shown are Emr1 (F4/80) and Cd68 (L), Itgax (M), and Ccl2, Ccr2, Tnf, and Il1b (N) (n = 8). Data represent means + SEM. *, P < 0.05; **, P < 0.01, ***, P < 0.001.
FIG 13
FIG 13
Wtap+/ mice exhibited better glucose tolerance and insulin sensitivity. (A to C) ITT (A), OGTT (B), and plasma insulin from the OGTT (C) in 16-week-old mice fed on NCD (n = 7 to 14). (D to F) ITT (D), OGTT (E), and plasma insulin from the OGTT (F) in 16-week-old mice fed on HFD for 8 weeks (n = 4 to 9). (G) Hyperinsulinemic-euglycemic clamp study on 28- to 29-week-old mice fed on HFD for 20 to 21 weeks. GIR, glucose infusion rate; HGP, hepatic glucose production; Rd, rate of glucose disappearance (n = 5 or 6). (H and I) Insulin signaling pathway studies. Phosphorylation of insulin receptor β-subunit (IRβ), insulin receptor substrate (IRS-1 and IRS-2), and Akt induced by a bolus injection of insulin was assessed in skeletal muscles (H) and livers (I). IP, immunoprecipitation. Data represent means ± SEM (A to F) or means + SEM (G to I). *, P < 0.05; **, P < 0.01; ***, P < 0.001.
FIG 14
FIG 14
Reduced adiposity in parenchymal tissues contributes to improvement of tissue inflammation and insulin sensitivity in Wtap+/− mice. Bone marrow transplantation (BMT) was performed on the indicated four groups, which represent BM from donor mice→recipient mice, in 34 week-old mice fed on HFD from 6 weeks after the transplantation for 20 weeks (n = 6 to 8). (A) Time course of the change in BW after BMT. (B) The fat mass as measured by DEXA. (C) The blood glucose levels in ITT (left) and its area under the curve (AUC) (right). (D to F) The gene expression of Emr1 and Cd68 (D), Itgax (E), and Tnf and Il1b (F) in eWAT. (G to I) The gene expression of Emr1 and Cd68 (G), Itgax (H), and Tnf and Il1b (I) in liver. (J) The weight of liver. Data represent means (A and C, left) and means + SEM (B; C, right; and D to J). *, P < 0.05 for WT→WT versus +/−→WT. P < 0.05 (§) and P < 0.01 (§§) for WT→WT versus WT→+/−. P < 0.05 (†) and P < 0.01 (††) for WT→WT versus +/−→+/−. P < 0.05 (¶) and P < 0.01 (¶¶) for +/−→WT versus WT→+/−. P < 0.05 (‡) and P < 0.01 (‡‡) for +/−→WT versus +/−→+/−.

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