Caspase 3 cleavage of Pax7 inhibits self-renewal of satellite cells

Abstract

Compensatory growth and regeneration of skeletal muscle is dependent on the resident stem cell population, satellite cells (SCs). Self-renewal and maintenance of the SC niche is coordinated by the paired-box transcription factor Pax7, and yet continued expression of this protein inhibits the myoblast differentiation program. As such, the reduction or removal of Pax7 may denote a key prerequisite for SCs to abandon self-renewal and acquire differentiation competence. Here, we identify caspase 3 cleavage inactivation of Pax7 as a crucial step for terminating the self-renewal process. Inhibition of caspase 3 results in elevated Pax7 protein and SC self-renewal, whereas caspase activation leads to Pax7 cleavage and initiation of the myogenic differentiation program. Moreover, in vivo inhibition of caspase 3 activity leads to a profound disruption in skeletal muscle regeneration with an accumulation of SCs within the niche. We have also noted that casein kinase 2 (CK2)-directed phosphorylation of Pax7 attenuates caspase-directed cleavage. Together, these results demonstrate that SC fate is dependent on opposing posttranslational modifications of the Pax7 protein.

Keywords: Pax7; casein kinase 2; caspase; satellite cells; self-renewal.

Fig. 1.

Caspase 3 activity is required during early satellite cell fate decisions. (A) Fibers were stained for cleaved-caspase 3 (green), Pax7 (red), and DAPI (blue) at 48 and 72 h following isolation. Active caspase 3 was observed at a low frequency in fiber-associated SCs at 48 and 72 h but not at time 0 in quiescent SCs. (B) Fibers were cultured in z.DEVD.fmk (DEVD; 20 µM) (Right) or DMSO (control) (Left) for 3 d and stained for Pax7 (red), MyoD (green), and DAPI (blue). (Scale bars: 10 µm.) (C) Quantification of the number of SCs expressing each marker at 3 d postinjury (P.I.) expressed as a percentage of the total number of SCs (>30 fibers per treatment per mouse; n = 4 mice). (D) Quantification of the number of self-renewing SCs (Pax7⁺/MyoD⁻) at 2, 3, and 4 d postinjury (Left) and the total number of SCs per fiber (>30 fibers per treatment per time point per mouse; n = 4 mice) (Right). The percentage of SCs undergoing self-renewal was significantly increase in DEVD-treated fibers compared with control at 3 and 4 d postinjury (*P < 0.05). (E) Mouse TA muscle was injured with CTX and injected with AdGFP or Adp35 at 2 d postinjury. TAs were embedded and 10-µm-thick frozen sections were stained for immunofluorescence (IF) analysis (Fig. S1C), the minimal fiber Feret’s diameter was calculated for GFP⁺ fibers, and the frequency of fibers in each bin size was expressed as a percentage of total number of fibers counted (>100 fibers/mouse; n = 3 mice/condition; *P < 0.05 AdGFP vs. Adp35). (F) Representative IF of muscle sections stained for GFP (green), Syn4 (red), laminin (white), and DAPI (blue). (Scale bars: 100 µm.) (G) The number of GFP⁺/Syn4⁺ SCs were counted per field indicating an increase in the number of infected SCs in Adp35 infected muscle compared with AdGFP control (*P < 0.05; n = 3). (H) Western blot of whole TA lysate isolated from 3 and 7 d regenerating muscle injected with AdGFP or Adp35 at 1 d postinjury. (I) Densitometry results were quantitated from the Western blot (G) and averaged from three separate experiments. Pax7 levels were only found significantly increased in day 7 Adp35 mice compared with uninjured control (ANOVA, P < 0.05). Error bars ± SEM.

Fig. S1.

Caspase inhibition limits satellite cell differentiation and perturbs muscle regeneration. (A) IF of single fibers isolated from Myf5-Cre/Rosa-YFP mice cultured in 20 µM z.DEVD.fmk (Right) or DMSO (control) (Left) stained with Pax7 (red), GFP (Myf5-YFP) (green), and DAPI (blue). (Scale bars: 10 µm.) (B) Quantification of the number of SCs expressing each marker expressed as a percentage of the total number of SCs (Left) and total number of SCs per fiber (Right) (n = 4; ±SEM; *P < 0.05). (C) IF of 2-wk-regenerating TA muscle injected with adenovirus expressing the caspase inhibitor p35 containing an IRES-GFP (Adp35) or the IRES-GFP backbone alone (AdGFP) 2 d post-CTX–induced injury. Sections were stained for GFP (green), laminin (red), and DAPI (blue). (Scale bars: 50 µm.) (D) H&E staining of 2-wk-regenerating TA muscle injected with either AdGFP or Adp35 2 d post-CTX–induced injury. (Scale bars: 200 µm.)

Fig. 2.

Pax7 protein is cleaved by caspase 3 at a cryptic cleavage site. (A) Differentiation time course of primary myoblasts treated with the caspase 3 peptide inhibitor z.DEVD.fmk (20 µM) or DMSO control. Lysates were probed for αPax7 (upper blots) and α-alpha-tubulin (lower blots; loading control). (B) Recombinant Pax7 protein and recombinant active caspase 3 were incubated for 3 h in standard cleavage assay conditions containing either DMSO or z.DEVD.fmk (20 µM) as indicated. Reactions were then subjected to SDS/PAGE and Western blot analysis using αPax7 indicating a caspase 3-specific cleavage event (ΔPax7). (C) Pax7 was subjected to caspase 3 cleavage as in B, followed by SDS/PAGE and silver stained using silver nitrate. Arrows indicate the Pax7 cleavage fragments (corresponding to ∼40 and ∼20 kDa). (D) Recombinant Pax7 protein containing aspartic acid to alanine point mutations at site D187 and D202 or D187 and D208 were subjected to caspase 3 cleavage, followed by Western blot analysis. Pax7 containing D187A/D202A was cleaved; however, D187A/D208A was completely blocked compared with wild type. (E) Luciferase Assay performed in COS cells cotransfected with a Pax7 containing plasmid and a luciferase reporter plasmid containing the Myf5 promoter. Error bars ± SEM; n = 3.

Fig. S2.

Analysis of the caspase 3 cleavage site within the Pax7 amino acid sequence. (A) The aspartic acid residues targeted by caspase 3 (highlighted in blue), as well as the serine residue targeted by CK2 (highlighted in red), are conserved across a broad range of phyla. Amino acid sequences were obtained from the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov). (B) Recombinant Pax7 was subjected to an in vitro caspase 3 cleavage reaction, followed by SDS/PAGE and silver stain as in Fig. 2C. The protein fragments produced were isolated and processed for LC-MS/MS. The green peptides represent those identified from the 20-kDa fragment (Fig. 2C, lower arrow) and the red peptides represents those identified from the 40-kDa fragment (Fig. 2C, upper arrow). (C) Recombinant Pax7 harboring single point mutations, altering aspartic acids D187, D202, and D208 to alanine, were subjected to in vitro cleavage assays with caspase 3 (Casp3) with or without the caspase inhibitor (z.DEVD.fmk), followed by SDS/PAGE and Western blot detection using an antibody against Pax7. Pax7 single mutants failed to fully block caspase 3-mediated cleavage, as assessed by the presence of a cleavage fragment (ΔPax7).

Fig. 3.

Small molecule (PAC-1) activation of caspase 3 induces cleavage of Pax7 and loss of SC self-renewal. (A) Single fibers were treated with either 50 µM PAC-1 or DMSO 60 h postisolation for 3 h, washed with fresh fiber media, and left for an additional 9 h. At 72 h postisolation, fibers were fixed and stained for Pax7 (red), Myogenin (green), and DAPI (blue). (Scale bars: 20 µm.) (B) The number of SCs expressing each marker was counted and the number of myogenin positive SCs (Pax7⁻/Myogenin⁺) was expressed as a percentage of total number of SCs (>30 fibers per treatment per mouse; n = 3 mice; *P < 0.05). (C) Western blot analysis of primary myoblasts treated with PAC-1 at the indicated concentrations for 24 h or differentiation media (Diff). Lysates were probed with αPax7, αCleaved-Caspase3, and αTubulin. (D) Densitometry analysis of Pax7 protein levels normalized to tubulin (loading control) shows a decrease similar to that seen in differentiation conditions (n = 3). (E) Primary myoblasts treated with 25 µM or 50 µM PAC-1, DMSO (control) or differentiation media (diff) for 24 h. RT-quantitative PCR (RT-qPCR) analysis using primers to Myf5 show a decrease in Myf5 expression in all treatment groups compared with DMSO control (n = 3; *P < 0.05; **P < 0.005). Error bars ± SEM.

Fig. S3.

Exogenous caspase activation promotes loss of Pax7 positive SCs. (A) Fibers were treated with either PAC-1 (50 µM) or DMSO 48 h following isolation for 24 h. At 72 h postisolation, fibers were fixed and stained with Pax7 (red), Syndecan 4 (Syn4) (green), and DAPI (blue). (Scale bars: 10 µm.) (B) The number of SCs expressing each marker was counted and expressed as a percentage of total number of SCs (n = 3; ±SEM; *P < 0.05). (C) Experimental conditions used for PAC-1 treatment did not induce a significant amount of cell death. Viability of primary myoblasts, following PAC-1 treatment for 24 h, were evaluated by PI staining and FACS analysis. Percentage of PI-negative cells were plotted for each treatment group (n = 3; ±SEM; *P < 0.005 relative to DMSO).

Fig. 4.

CK2 phosphorylates Pax7 and restricts SC differentiation. (A) Fibers were stained for CK2α (green), Pax7 (red), and DAPI (blue) (Left). CK2 is expressed by fiber associated SCs at all time points tested (n = 3) (Right). (B) Fibers were cultured in the presence of the CK2 inhibitor TBBt (50 µM) or DMSO for 3 d and stained for Pax7 (red), MyoD (green), and DAPI (blue). (C) Quantification of the number of SCs expressing each marker expressed as a percentage of total SCs. Fibers treated with TBBt contained more differentiating Pax7⁻/MyoD⁺ SCs compared with DMSO control (n = 4; *P < 0.05). (D) Schematic of Pax7 protein indicating the caspase 3 cleavage sites, as well as the CK2 phosphorylation site located between the paired domain (PD) and the homeodomain (HD) of Pax7. The octapeptide (OP) sequence is bolded. (E) In vitro kinase assay of CK2 and Pax7. Autoradiography indicates Pax7 is phosphorylated by CK2 in a dose-dependent manner. (F) In vitro kinase assay of wild-type Pax7 or Pax7 with site mutations at S201A or S205A. Autoradiography indicating loss of phosphorylation in the S201A mutant only. (G) Recombinant Pax7 was subjected to an in vitro kinase assay as in E. Aliquots from each reaction were then subjected to caspase 3 cleavage, SDS/PAGE, and Western blot analysis using αPax7. Production of the Pax7 cleavage fragments (ΔPax7, † and ‡) was impaired in the D187A mutant following preincubation with CK2. (H, Left) Primary myoblasts were fixed at the indicated times and stained for Pax7 (red) (Top), CK2 (green) (Middle), or PLA [red dots indicate PLA reaction, costained with DAPI (blue)] (Bottom). (H, Right) PLA showed a positive interaction between CK2 and Pax7 at growth and 6 h following differentiation (6 h Diff). The number of PLA-positive puncta per cell was quantified (80–150 cells/treatment; n = 3). Error bars represent ± SEM. *P < 0.05. (Scale bars: 10 µm.)

Fig. S4.

CK2 phosphorylates and interacts with Pax7. (A) Kinase activity of CK2 on Pax7 was completely inhibited by the CK2 small molecule inhibitor TBBt. Recombinant Pax7 was subjected to an in vitro kinase assay with purified GST-CK2, in the presence or absence of TBBt (50 µM), followed by SDS/PAGE and autoradiography. (B) Pax7 is phosphorylated by CK2 at serine 201. Dephosphorylated Pax7 was subjected to a cold in vitro kinase assay with (or without) GST-CK2, followed by SDS/PAGE and silver stain (Left). The bands were excised and processed for LC-MS/MS. Phosphorylation on serine 201 was identified in the +CK2 sample only (Right, lower graph). (C) Fibers were fixed at 48 h postisolation and stained using the PLA assay. Red dots indicated an interaction between Pax7 and CK2. Fibers were costained with Rat anti-α7-integrin (white) and DAPI (blue). (Scale bar: 10 µm.)