Nuclear Translocation of PFKFB3 Promotes Disuse‐Induced Muscle Atrophy via Scaffolding Nedd4‐Mediated JunB Ubiquitination

Figures

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  FIGURE 1. PFKFB3 is upregulated in disuse‐induced muscle atrophy. (A) Schematic summary of non‐targeted metabolomics analysis of tibialis anterior (TA) muscles from disuse‐induced muscle atrophy mice and control mice. Quantification bar plots are presented as mean ± SD, showing raw intensity values per biological replicate. Paired samples are connected by black lines. Differentially abundant metabolites are indicated as colored dots. n = 6. Adjusted p values were calculated using paired two‐tailed Student's t‐test or Wilcoxon test. NS, not significant; ND, not detected. (B) KEGG pathway enrichment analysis of differentially expressed genes from the GSE211204 dataset (log2FC (IM/Control) ≥ 1, p < 0.05). (C) Heatmap of differentially expressed genes involved in the glycolysis pathway. (D) Schematic diagram of the disuse‐induced muscle atrophy mouse model. The schematic diagram was generated by biorender. (E) Measurement of hindlimb grip strength in control and atrophy mice. (F) Representative images of TA muscles from control and atrophy mice (n = 12). (G) Quantification of TA muscle mass. (H–I) Hematoxylin and eosin (H&E) staining of TA muscle sections (H) and quantification of myofiber…

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  FIGURE 2. Muscle‐specific overexpression of PFKFB3 induces and exacerbates muscle atrophy. (A) Schematic diagram of AAV‐mediated overexpression of PFKFB3 in mouse tibialis anterior muscle (TA) (n = 5). The schematic diagram was generated by biorender. (B) The grip strength measurements of the mice in the control group (without AAV), AAV‐Mock, AAV‐oePFKFB3, IM + AAV‐Mock, and IM + AAV‐oePFKFB3 groups were conducted. (C) The tibialis anterior muscles of the control group (without AAV), AAV‐Mock, AAV‐oePFKFB3, IM + AAV‐Mock, and IM + AAV‐oePFKFB3 were examined. (D) The measurements of the mass of the anterior tibialis muscle in the control group (without AAV), the AAV‐Mock group, the AAV‐oePFKFB3 group, the IM + AAV‐Mock group, and the IM + AAV‐oePFKFB3 group. (E–G) H&E staining of TA muscle sections (E) and quantification of myofiber CSA (F‐G). (H, I) The protein expression levels of PFKFB3, MuRF1, and Atrogin‐1(H), along with their quantitative analyses (I), across the five experimental groups: Control (no AAV), AAV‐Mock, AAV‐oePFKFB3, IM + AAV‐Mock, and IM + AAV‐oePFKFB3. Original blot can be found in Figure S12E–H. All data are presented as mean ± SEM. P values: *p < 0.05, **p < 0.01,…

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  FIGURE 3. PFKFB3 induces atrophy in C2C12 myotubes. (A) Immunofluorescence staining of C2C12 myotubes overexpressing PFKFB3. (B) Quantification of myotube length and fusion index compared to control. (C) Immunofluorescence staining of C2C12 myotubes with PFKFB3 knockdown. (D) Quantification of myotube length and fusion index. (E, F) mRNA levels of MyHC, MuRF1, and Atrogin‐1 in PFKFB3‐overexpressing (E) and knockdown (F) myotubes. (G–J) Protein levels of MyHC, MuRF1, and Atrogin‐1 in PFKFB3‐overexpressing (G, H) and knockdown (I, J) groups. Original blot can be found in Figure S12I–M and Figure S13A–E. (K, L) Immunofluorescence staining of MyHC (K) and quantification of myotube length (L) in 3‐PO‐treated vs. PFKFB3‐overexpressing myotubes. All data are presented as mean ± SEM. P values: *p < 0.05, **p < 0.01, ***p < 0.001.

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  FIGURE 4. Nuclear PFKFB3 predominantly mediates myotube atrophy. (A) Immunofluorescence localization of PFKFB3 in C2C12 cells. (B) Nuclear and cytoplasmic fractionation of C2C12 cells. Original blot can be found in Figure S13F–H. (C) Schematic of the canonical nuclear localization sequence (NLS) and NLS‐mutant constructs of PFKFB3. (D) Expression levels of PFKFB3 in different C2C12 models. Original blot can be found in Figure S13I, J. (E) The results of immunofluorescence staining for PFKFB3 in C2C12 cells under each experimental condition. (F) Nuclear–cytoplasmic fractionation and Western blot analysis in WT+NLS and K501Q groups. Original blot can be found in Figure S13K–P. (G, H) MyHC immunofluorescence staining (G) and quantification of myotube length (H).

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  FIGURE 5. PFKFB3 regulates muscle atrophy via the transcription factor JunB. (A) Venn diagram showing overlap between proteins identified by IP‐MS in IP and IgG groups. (B, C) MyHC immunofluorescence staining (B) and quantification of myotube length and fusion index (C) upon JunB overexpression. (D) Co‐immunoprecipitation (Co‐IP) showing interaction between PFKFB3 and JunB. Original blot can be found in Figure S14A–D. (E, F) Confocal immunofluorescence colocalization (E) and correlation analysis (F). (G) GST pull‐down assay demonstrating direct interaction. Original blot can be found in Figure S14E, F. (H, I) Effects of PFKFB3 overexpression or knockdown on JunB protein levels. Original blot can be found in Figure S14G–L. (J, K) MyHC staining (J) and myotube length quantification (K).

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  FIGURE 6. PFKFB3 promotes ubiquitin‐mediated degradation of JunB. (A) The effects of overexpression of PFKFB3 and knockdown of PFKFB3 on the RNA level of JunB. (B, C) Cycloheximide (CHX) chase assay and quantification in PFKFB3‐overexpressing cells. Original blot can be found in Figure S14M–P. (D, E) CHX assay and quantification in PFKFB3 knockdown cells. Original blot can be found in Figure S15A‐D. (F, G) Western blot and quantification of JunB protein levels after treatment with MG132 or chloroquine (CQ). Original blot can be found in Figure S15E–G. (H) Using the antibody of JunB for IP experiments, combined with the JunB protein, the ubiquitin levels of JunB were respectively detected when PFKFB3 was overexpressed or knocked down. Original blot can be found in Figure S15H–O. All data are presented as mean ± SEM. NS, not significant; *p < 0.05, **p < 0.01, ***p < 0.001.

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  FIGURE 7. Nedd4 is identified as an E3 ubiquitin ligase for JunB. (A) Prediction of JunB E3 ligases using the UbiBrowser database. (B, C) In C2C12 cells, the antibody of JunB was used as the IP antibody to detect the protein level of Nedd4. The Nedd4 antibody was used as an IP antibody to detect the protein level of JunB. Original blot can be found in Figure S16A–D. (D–E) Immunofluorescence staining was performed simultaneously on Nedd4 and JunB in C2C12 cells, and co‐localization and related analysis were conducted. (F) GST pull‐down assay. The GST‐tagged JunB protein was expressed in Escherichia coli and purified using glutathione‐S‐ubiquitin resin. The cell lysates containing potential interacting proteins were incubated with the immobilized GST fusion protein. Original blot can be found in Figure S16E, F. (G, H) JunB protein levels and quantification upon Nedd4 overexpression or knockdown. Original blot can be found in Figure S16G‐I. (I) Flag‐JunB and HA‐Ub were respectively co‐transfected into 293T cells. An IP experiment was conducted using Flag antibodies to detect the levels of HA in the overexpressed Nedd4 group and the control group. Original blot can be found in Figure S16J–N.…

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  FIGURE 8. PFKFB3 acts as a scaffold to facilitate Nedd4–JunB interaction. (A) Co‐IP assays showing interactions among PFKFB3, Nedd4, and JunB. Original blot can be found in Figure S17B–E. (B) GST pull‐down assay between PFKFB3 and Nedd4. Original blot can be found in Figure S17F, G. (C, D) Confocal colocalization and correlation analysis. (E) Effect of PFKFB3 expression on Nedd4–JunB interaction. Original blot can be found in Figure S17H–O. (F) Effects of PFKFB3 or Nedd4 knockdown on JunB ubiquitination. Original blot can be found in Figure S17P‐U. (G) Effects of JunB on PFKFB3 mRNA levels. (H) Schematic of predicted JunB‐binding sites in the PFKFB3 promoter and mutant constructs. (I) Luciferase reporter assay for PFKFB3 promoter activation by JunB. (J, K) ChIP‐PCR (J) and qPCR quantification (K) validating JunB binding. All data are presented as mean ± SEM. P values: *p < 0.05, **p < 0.01, ***p < 0.001.

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  FIGURE 9. Targeting the PFKFB3–Nedd4–JunB axis alleviates muscle atrophy. (A) Immunohistochemical staining of PFKFB3, Nedd4, and JunB in muscle tissues (scale bar, 100 µm). (B, C) Correlation analyses between PFKFB3 and Nedd4 (B), and PFKFB3 and JunB (C). (D) Representative TA muscle images following treatment with 3‐PO, XMU‐MP‐10, or combination. (E, F) Grip strength (E) and TA muscle mass (F). (G) H&E staining of muscle sections. (H) Quantification of CSA. All data are presented as mean ± SEM. P values: *p < 0.05, **p < 0.01, ***p < 0.001.