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. 2021 Dec;12(6):1776-1788.
doi: 10.1002/jcsm.12774. Epub 2021 Aug 24.

p21-activated kinase 4 phosphorylates peroxisome proliferator-activated receptor Υ and suppresses skeletal muscle regeneration

Yuancheng Mao  1 , Chang Yeob Han  2 , Lihua Hao  1 , In Hyuk Bang  1 , Eun Ju Bae  2 , Byung-Hyun Park  1
Affiliations

p21-activated kinase 4 phosphorylates peroxisome proliferator-activated receptor Υ and suppresses skeletal muscle regeneration

Yuancheng Mao et al. J Cachexia Sarcopenia Muscle. 2021 Dec.

Abstract

Background: Skeletal muscle regeneration is an adaptive response to injury that is crucial to the maintenance of muscle mass and function. A p21-activated kinase 4 (PAK4) serine/threonine kinase is critical to the regulation of cytoskeletal changes, cell proliferation, and growth. However, PAK4's role in myoblast differentiation and regenerative myogenesis remains to be determined.

Methods: We used a mouse model of myotoxin (notexin)-induced muscle regeneration. In vitro myogenesis was performed in the C2C12 myoblast cell line, primary myoblasts, and primary satellite cells. In vivo overexpression of PAK4 or kinase-inactive mutant PAK4S474A was conducted in skeletal muscle to examine PAK4's kinase-dependent effect on muscle regeneration. The regeneration process was evaluated by determining the number and size of multinucleated myofibres and expression patterns of myogenin and eMyHC. To explore whether PAK4 inhibition improves muscle regeneration, mice were injected intramuscularly with siRNA that targeted PAK4 or orally administered with a chemical inhibitor of PAK4.

Results: p21-activated kinase 4 was highly expressed during the myoblast stage, but expression gradually and substantially decreased as myoblasts differentiated into myotubes. PAK4 overexpression, but not kinase-inactive mutant PAK4S474A overexpression, significantly impeded myoblast fusion and MyHC-positive myotube formation in C2C12 cells, primary myoblasts, and satellite cells (P < 0.01). Conversely, PAK4 silencing led to an 8.7% and a 20.3% increase in the number of multinucleated larger myotubes in C2C12 cells and primary myoblasts. Further, in vivo overexpression of PAK4 by adenovirus injection to mice prior to and after myotoxin-induced injury led to a 52.6% decrease in the number of eMyHC-positive myofibres on Day 5 in tibialis anterior muscles as compared with those injected with control adenoviruses (P < 0.01), while Ad-PAK4S474A showed comparable muscle regeneration parameters. PAK4-induced repression of muscle regeneration coincided with an increase in phosphatase and tensin homologue (PTEN) expression and a decrease in phosphoinositide 3-kinase-Akt signalling. In contrast, PAK4 silencing reduced PTEN expression in mice. Consistent with these findings, prodrug of PAK4 inhibitor CZh-226 (30 mg/kg) orally administered to mice repressed PTEN expression and accelerated myotube formation. Subsequent mechanistic studies revealed that PAK4 directly phosphorylates PPARγ at S273 to increase its transcription activity, thereby up-regulating PTEN expression. Importantly, an analysis of the Genotype-Tissue Expression database showed a positive correlation between PAK4 and PTEN in human skeletal muscle tissues (P < 0.01).

Conclusions: p1-activated kinase 4 is a new member of PPARγ kinase, and PAK4 inhibition may have a therapeutic role as an accelerant of muscle regeneration.

Keywords: Muscle regeneration; Myogenesis; PAK4; PPARγ; PTEN.

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Figures

Figure 1
Figure 1
Suppression of myogenesis in PAK4‐overexpressing C2C12 cells and primary myoblasts. C2C12 myoblasts (A–C) and primary myoblasts (D–F) were infected with adenoviruses expressing β‐galactosidase (LacZ), wild‐type PAK4, or a PAK4 kinase‐inactive S474A mutant and differentiated for 5 days. (A, D) Cells were immunostained with anti‐MyHC antibody. Myogenic conversion was scored by quantifying the fusion index and myotube diameter. (B, E) Protein levels of myogenic markers and (C, F) mRNA levels of myosin heavy chains and myogenin were determined by western blotting and qPCR, respectively. Values are mean ± SD. * P < 0.05 and ** P < 0.01 vs. Ad‐LacZ; # P < 0.05 and ## P < 0.01 vs. Ad‐PAK4.
Figure 2
Figure 2
Suppression of myogenesis in PAK4‐overexpressing satellite cells (SCs). (A–C) Immunofluorescence staining and western blotting analysis for PAK4 in SCs cultured in growth medium for different time periods (from G0 to G4) followed by 4 day culture in differentiation medium (D4). (D, E) SCs were infected with PAK4 and mutant PAK4 adenoviruses and cultured in growth medium (GM) for 6 h and in differentiation medium (DM) for another 4 days. Protein levels of myogenic markers (MyHC and Myog) and myotube formation were determined. Values are mean ± SD. ** P < 0.01 vs. Ad‐LacZ; ## P < 0.01 vs. Ad‐PAK4.
Figure 3
Figure 3
Impairment of skeletal muscle regeneration in PAK4‐overexpressing mice. (A) The tibialis anterior (TA) muscles of C57BL/6 mice were injected with Ad‐LacZ, Ad‐PAK4, or Ad‐PAK4S474A and then injured by intramuscular injection of NTX. (B) Time course analysis of PAK4 and myogenic markers after NTX injection by western blotting. (C) Immunofluorescence analysis of eMyHC‐positive fibres in TA muscles. (D) Immunofluorescence staining of Myog‐positive or desmin‐positive myofibres at Day 5. Arrowheads indicate MyoG‐positive myofibres. (E) Western blot analysis of Myog and eMyHC in injured TA muscles of control and PAK4‐overexpressing mice at 3 or 5 days after injury (n = 3). (F) H&E and immunofluorescence analyses of sections. Average cross‐sectional area (CSA) of regenerating myofibres and the percentage of myofibres containing two or more centrally located nuclei per field were determined from immunofluorescence sections. Values are mean ± SD. * P < 0.05 and ** P < 0.01 vs. Ad‐LacZ; # P < 0.05 and ## P < 0.01 vs. AdPAK4.
Figure 4
Figure 4
Acceleration of skeletal muscle regeneration in PAK4‐silenced mice. The tibialis anterior (TA) muscles of C57BL/6 mice were injected with scrambled siRNA (siCtrl) or siRNA against PAK4 (siPAK4) and then injured by intramuscular injection of NTX as shown in Figure 3A. (A) Western blotting analysis for PAK4 in PAK4 silenced muscles. (B) Immunofluorescence analysis of eMyHC‐positive fibres in TA muscles. (C) H&E and immunofluorescence analyses of sections. Average cross‐sectional area (CSA) of regenerating myofibres and the percentage of myofibres containing two or more centrally located nuclei per field were determined from immunofluorescence sections. (D) Time course analysis of myogenic markers by western blotting. (E) Immunofluorescence staining of Myog‐positive or desmin‐positive myofibres at Day 5. Arrowheads indicate MyoG‐positive myofibres. Values are mean ± SD. ** P < 0.01 vs. D0; ## P < 0.01 vs. siCtrl.
Figure 5
Figure 5
Suppression of PI3K‐Akt pathway by PAK4. (A, B) Pak4 gene was either overexpressed or silenced in C2C12 cells as indicated, and PI3K‐Akt signalling pathway on Day 5 was analysed by western blotting. (C, D) The tibialis anterior (TA) muscle was injected with either PAK4 adenovirus or PAK4 siRNA and PI3K‐Akt signalling pathway on 7 days after NTX injection was analysed by western blotting. (E) mRNA levels of Pten were determined in PAK4‐overexpressing C2C12 cells and TA muscles. (F, G) C2C12 myoblasts were transfected with siCtrl or siPTEN, and PI3K‐Akt signalling and myotube formation were compared. Values are mean ± SD. ** P < 0.01 vs. Ad‐LacZ or siCtrl; ## P < 0.01 vs. AdPAK4 or siCtrl + PAK4.
Figure 6
Figure 6
Increase of PPARγ‐mediated PTEN transcription by PAK4. (A) Genotype‐Tissue Expression (GTEx) analysis of human skeletal muscle. Pearson correlation coefficients between PAK4 and PTEN, PAK4 and PPARG, and PPARG and PTEN in human skeletal muscle were calculated. TPM, transcripts per million. (B, C) After transfection of HEK293T cells as indicated, PPRE‐luciferase and PTEN‐luciferase activities were determined. (D) Maps of human and mouse Pten promoters and ChIP‐qPCR assay showing binding of PPARγ to the Pten promoters. (E, F) C2C12 myoblasts were transfected with scrambled siRNA (siCtrl) or siRNA against PPARγ (siPPARγ), and protein levels of PTEN and PI3K‐Akt pathway and myotube formation were compared. Values are mean ± SD. ** P < 0.01 vs. none, IgG, or siCtrl; ## P < 0.01 vs. PPARγ, Ad‐LacZ, or siCtrl + PAK4; $$ P < 0.01 vs. PAK4 + PPARγ or Ad‐PAK4.
Figure 7
Figure 7
Direct phosphorylation of PPARγ by PAK4. (A) Recombinant PPARγ was incubated with active PAK4 and [32P]ATP for 30 min at 30°C, and proteins in the mixture were resolved by SDS‐PAGE. The band was visualized by autoradiography of 32P‐labelled protein. Loading of proteins was confirmed by Coomassie blue staining. (B, C) After transfection of HEK293T cells as indicated, co‐IP was performed to determine PAK4 interaction with and phosphorylation of PPARγ. (D) C2C12 myoblasts were PAK4 overexpressed or silenced, and then phosphorylation of PPARγ, ERK, and CDK5 on Day 5 was analysed by western blotting. (E) C2C12 myoblasts were transfected with scrambled siRNA (siCtrl) or siRNA against CDK5 (siCDK5), and then PAK4 phosphorylation of PPARγ was analysed by western blotting. (F) C2C12 myoblasts were pretreated with ERK inhibitor PD98059 (10 μM), and PAK4 phosphorylation of PPARγ was analysed. (G) PTEN‐luciferase activities were determined after transfection of HEK293T cells as indicated. (H) C2C12 cells were transfected with wild‐type or mutant PPARγ (S273A) along with PAK4, and then PI3K–Akt signalling and myogenic markers on Day 5 were determined. Values are mean ± SD. ** P < 0.01 vs. none; ## P < 0.01 vs. PPARγ; $$ P < 0.01 vs. PAK4 + PPARγ.
Figure 8
Figure 8
Acceleration of skeletal muscle regeneration by a small molecule inhibitor of PAK4. (A) C57BL/6 mice were treated with prodrug of PAK4 inhibitor CZh‐226‐P (30 mg/kg) via oral gavage 2 days before NTX injection and every day before and after NTX injection until Day 13. (B) Serum levels of AST and ALT were measured. (C) Western blot analysis of PPARγ–PTEN–Akt signalling and myogenic markers in injured TA muscles at 5 days after injury. (D) H&E and immunofluorescence analyses at 5 or 14 days after injury. Average cross‐sectional area (CSA) of regenerating myofibres and the percentage of myofibres containing two or more centrally located nuclei per field were determined from immunofluorescence sections. (E) The tibialis anterior (TA) muscles of C57BL/6 mice were injected with siCtrl or siPAK4 and then injured by intramuscular injection of NTX. CZh‐226‐P (30 mg/kg) was administered once a day via oral gavage for 7 days starting 2 days before NTX injection. Myog (+) and eMyHC (+) fibres in TA muscles were counted at Day 5. Values are mean ± SD. ** P < 0.01 vs. vehicle. CZh‐226‐P, prodrug of CZh‐226.
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