Author Response
eLife assessment
This manuscript provides useful information about the lipid metabolite 15d-PGJ2 as a potential regulator of myoblast senescence. The authors provide experimental evidence that 15d-PGJ2 inhibits myoblast proliferation and differentiation by binding and regulating HRas. However, the manuscript is incomplete in its current form, as it lacks robust support from the data regarding the main conclusions related to senescence and technical concerns related to the senescence models used in this study.
Authors Response- We ae grateful to the editors and the reviewers for their time and comments in sharpening the science and the writing of the manuscript. We have attached a detailed response to emphasize that the manuscript does include robust evidence regarding the claims, which could have been missed during the review process. We have provided a better context for these points now.
Public Reviews:
Reviewer #1 (Public Review):
Summary:
The authors show that upon treatment with Doxorubicin (Doxo), there is an increase in senescence and inflammatory markers in the muscles. They also show these genes get upregulated in C2C12 myoblasts when treated with conditioned media or 15d-PGJ2. 15dPGJ2 induces cell death in the myoblasts, decreases proliferation (measured by cell numbers), and decreases differentiation and fusion. 15d-PGJ2 modified Cys184 of HRas, which is required for its activation as indicated by the FRET analysis with RAF RBD. They also showed that 15d-PGJ2 activates ERK signaling, but not Akt signaling, through the electrophilic center. 15d-PGJ2 inhibits Golgi localization of HRAS (only WT, not C181 or C184 mutant). They also showed that expressing the WT HRas followed by 15d-PGJ2 treatment led to a decrease in the levels of MHC mRNA and protein, and this defect is dependent on C184. This is a well-written manuscript with interesting insights into the mechanism of action of 15d-PGJ2. However, some clarification and experiments will help the paper advance the field significantly.
Strengths:
The data clearly shows that 15d-PGJ2 has a negative role in the myoblast cells and that it leads to modification of HRas protein. Moreover, the induction of biosynthetic enzymes in the PGD2 pathway also supports the induction of 15d-PGJ2 in Doxorubicin-treated cells. Both conditioned media experiments and the 15d-PGJ2 experiments show that 15d-PGJ2 could be the active component secreted by the senescent myoblasts.
Weaknesses:
The genes that are upregulated in the muscles upon injection with Doxo are also markers for inflammation. Since Doxo is also known to induce systemic inflammation, it is important to delineate these two effects (inflammatory cells vs senescent cells). The expression of beta Gal and other markers of senescence in the tissue sections will help to delineate these.
As pointed out Doxo induces systemic inflammation along with inducing DNA damage-mediated senescence. Therefore, along with the inflammatory markers of the SASP (CXCL1/2, TNF1α, IL6, PTGS1/2, PTGDS) we also observed an increase in the mRNA levels of canonical markers of DNA damage-mediated senescence. We observed an increase in the mRNA levels of cell cycle and senescence associated proteins p16 and p21 (Fig. 1C). We also observed an increased nuclear accumulation of p21 (Fig. 1A) and increased levels of phosphorylated H2A.X in the nucleus (Fig. 1B). We will characterize other markers of senescence including senescence-associated β galactosidase in the revised manuscript.
In Figure 2, where the defect in the differentiation of myoblasts upon treatment with 15d-PGJ2 is shown, most of the cells die within 48 hours at higher concentrations, making it difficult to perform the experiments. This also shows that 15d-PGJ2 was toxic to these cells. Lower concentrations show a decrease in the differentiation based on the lower number of nuclei in fibers and low expression of MyoD, MyoG, and MHC. However, it is unclear if this is due to increased cell death or defective differentiation. It would be a lot more informative if the cell count, cell division, and cell death could be plotted for these concentrations of the drug during the experiment.
We only observed the death of cells at higher concentrations of 15d-PGJ2 (5 µM and 10 µM) (Fig. S2A), but not significantly at the 4 µM concentration used in Figure 2. This is the reason 4uM was used, and we should have clarified this. We will include viability data for the low concentration of 15d-PGJ2 (4 µM) in the revised manuscript.
Also, in the myoblast experiments, are the effects of treatment with Dox reversible?
The treatment with Doxorubicin is irreversible as the senescent phenotype was not reversed after withdrawal of Doxorubicin, even after 20 days.
In Figure 3, most of the experiments are done at a high concentration, which induces almost complete cell death within 48 hours.
Figure 3 is an acute experiment for only 1 hour, at which time no cell death was observed. Specifically, we measured the phosphorylation of Erk and Akt proteins after 1 hour of treatment with 15d-PGJ2 (10 µM) during which we did not observe any cell death.
Even at such a high concentration of 15dPGJ2, the increase in ERK phosphorylation is minimal.
We observe a ~30% increase in the phosphorylation of Erk proteins after treatment with 15d-PGJ¬2 in 0.2% serum medium compared to treatment with vehicle (DMSO). This is reproducible and significant.
The experiment Figure 4C shows that C181 and C84 mutants of the HRas show higher levels in Golgi compared with WT. However, this could very well be due to the defect in palmitoylation rather than the modification with 15d-PGJ2.
Our data does not suggest higher levels of C184S mutant in the Golgi compared with WT (Fig. S4A). We observed that the ratio of HRas levels in the Golgi to the HRas levels in the plasma membrane were similar in C2C12 cells expressing HRas C184S and HRas WT (Fig. S4A graph columns 1 and 5).
Though the authors allude to the possibility that intracellular redistribution of HRas by 15d-PGJ2 requires C181 palmitoylation, the direct influence of C184 modification on C181 palmitoylation is not shown. To have a meaningful conclusion, the authors need to compare the palmitoylation and modification with 15d-PGJ2.
Palmitoylation of HRas C181S is required for the localization of HRas at the plasma membrane. The inhibition of palmitoylation of C181, either by mutation (C181S) or treatment with protein palmitoyl transferase inhibitor (2-Bromopalmitate), results in the accumulation of HRas at Golgi(Rocks et al., 2005) (Fig. S4A). Modification of HRas at C184 by 15d-PGJ2 (Fig. 3A) could inhibit the palmitoylation of HRas at C181. However, our data does not support this hypothesis as modification of HRas WT by 15d-PGJ2 does not increase the level of HRas at the Golgi, like in the case of inhibition of cysteine palmitoylation due to C181S mutation.
To test if the inhibition of myoblast differentiation depends on HRas, they overexpressed the HRas and mutants in the C2C12 lines. However, this experiment does not take the endogenous HRAs into consideration, especially when interpreting the C184 mutant. An appropriate experiment to test this would be to knock down or knock out HRas (or make knock-in mutations of C184) and show that the effect of 15d-PGJ2 disappears.
Endogenous HRas (wild type) is present in the C2C12 cells overexpressing the EGFP-tagged HRas constructs. Therefore, we only observe a partial rescue in the differentiation after 15d-PGJ2 treatment in C2C12 cells expressing the C184S mutant (Fig. 4D and E). However, since HRas is expressed under high expression CMV promoter and in the absence of other regulatory elements, the overexpressed constructs do show a dominant effect over the endogenous HRas, showing cysteine mutant dependent inhibition of differentiation of myoblasts after treatment with 15d-PGJ2 (Fig. 4D and E).
Moreover, in this specific experiment, it is difficult to interpret without a control with no HRas construct and another without the 15d-PGJ2 treatment.
The mRNA levels of MyoD, MyoG, and MHC in C2C12 cells expressing HRas constructs after treatment with 15d-PGJ2 were normalized to the mRNA levels in C2C12 cells expressing corresponding constructs and were treated with vehicle (DMSO). mRNA levels in C2C12 cells treated with vehicle were not shown as they were normalized to 1. MHC protein levels in C2C12 cells expressing HRas constructs after 15d-PGJ2 treatment were normalized to that in C2C12 cells treated with vehicle (DMSO). Since the hypothesis to study the effect of HRas cysteine mutations on the differentiation of myoblasts after treatment with 15d-PGJ2, C2C12 cells expressing HRas WT serve as adequate control. Fig. 2 shows the effect of 15d-PGJ2 on muscle differentiation when HRas was not overexpressed.
Moreover, the overall study does not delineate the toxic effects of 15d-PGJ2 from its effect on the differentiation.
The inhibition of differentiation in C212 cells after treatment with 15d-PGJ2 cannot be attributed to the general toxicity of 15d-PGJ2 in cells. We show that the inhibition of differentiation of myoblasts after 15d-PGJ2 depends on modification of HRas at C184 i.e. failure to modify HRas at C184 (Fig. 3A) and resultant activation (Fig. 3B) by 15d-PGJ2 rescues this inhibition of differentiation of C2C12 cells (Fig. 4D and E), dissecting the inhibition of differentiation of myoblasts by 15d-PGJ2 from general toxic effects of 15d-PGJ2 on cell physiology.
Please note that the effect of 15d-PGJ2 on cell physiology is context-specific. On one hand, 15d-PGJ2 has been shown to exert tumor-suppressor effects by inhibiting the proliferation of ovarian cancer cells and lung adenocarcinoma cells (de Jong et al., 2011; Slanovc et al., 2024), 15d-PGJ2 also exerts pro-carcinogenic effects by induction of epithelial to mesenchymal transition in breast cancer cells MCF7 and inhibition of tumor-suppressor protein p53 in MCF7 and PC-3 cells (Choi et al., 2020; Kim et al., 2010).
Reviewer #2 (Public Review):
Summary:
In this study, Swarang and colleagues identified the lipid metabolite 15d-PGJ2 as a potential component of senescent myoblasts. They proposed that 15d-PGJ2 inhibits myoblast proliferation and differentiation by binding and regulating HRas, suggesting its potential as a target for restoring muscle homeostasis post-chemotherapy.
Strengths:
The regulation of HRas by 15d-PGJ2 is well controlled.
Weaknesses:
The novelty of the study is compromised as the activation of PGD and 15d-PGJ2, as well as the regulation of HRas and cell proliferation, have been previously reported.
Literature does support this statement, and it is important to clarify this mis-impression for the field as whole
Let us clarify-
Covalent modification of HRas by 15d-PGJ2 has been reported only twice in the literature(Luis Oliva et al., 2003; Yamamoto et al., 2011) in fibroblasts and neurons respectively.
Interaction between HRas and 15d-PGJ2 in skeletal muscles has not been shown before, even though both HRas and 15d-PGJ2 are shown to be key regulators of muscle homeostasis.
Activation of HRas by 15d-PGJ2 was reported first by Luis Oliva et al (Luis Oliva et al., 2003). However, this study does not comment on the functional implications of activation of HRas signaling.
Recently, our lab contributed to a study where the functional implication of activation of HRas signaling due to covalent modification by 15d-PGJ2 was shown in the maintenance of senescence phenotype (Wiley et al., 2021).
15d-PGJ2 was shown to inhibit the differentiation of myoblasts by Hunter et al (Hunter et al., 2001). This study hypothesized that the inhibition of myoblast differentiation is via 15d-PGJ2 mediated activation of the PPARγ signaling, the study also showed inhibition of myoblast differentiation independent of PPARγ activity, suggesting the presence of other mechanisms.
This is the first study to show a molecular mechanism where activation of HRas signaling in skeletal myoblasts due to covalent modification by 15d-PGJ2 at C184 of HRas inhibits the differentiation of skeletal myoblasts.
Additionally, there are major technical concerns related to the senescence models, limiting data interpretation regarding the relevance to senescent cells.
Major concerns:
(1) The C2C12 cell line is not an ideal model for senescence study due to its immortalized nature and lack of normal p16 expression. A more suitable myoblasts model is recommended, with a more comprehensive characterization of senescence features.
C2C12 is a good model for DNA damage based senescence that is used in this manuscript. It is not a models for replicative senescence since it is immortalized. In this study we show that C2C12 cells undergo DNA damage mediated senescence after treatment with Doxo. We also observe similar phenotype in MCF7 breast cancer cells and IMR90 lung fibroblasts after treatment with Doxo (Data will be updated in the supplementary figure 1). Also, several reports in the literature have shown induction of senescence in C2C12 cells. Moiseeva et al 2023 show induction of senescence in C2C12 cells after etoposide mediated DNA damage. Moustogiannis et al 2021 show induction of replicative senescence in C2C12 cells.
(2) The source of increased PGD or its metabolites in the conditioned medium is unclear. Including other senescence models, such as replicative or oncogene-induced senescence, would strengthen the study.
Fig. 1E shows time dependent increase in the expression of PGD2 biosynthetic enzymes in senescent C2C12 cells. Fig. 1F shows increase in the levels of 15d-PGJ2 secreted by senescent C2C12 cells in the conditioned medium. This data shows that senescent C2C12 cells are the source of PGD and its metabolites in the conditioned medium.
Again, C2C12 is not suitable for replicative senescence due to its immortalized status.
We and others have shown that C2C12 cells undergo senescence, and this manuscript only used DNA damage induced senescence.
(3) In the in vivo part, it is unclear whether the increased expression of PTGS1, PTGS2, and PTGDS is due to senescence or other side effects of DOXO.
We concur that this is a limitation of this study and the subsequent work will demonstrate the origin of prostaglandin biosynthesis after treatment with Doxo in vivo.
(4) Figure 2A lacks an important control from non-senescent cells during the measurement of C2C12 differentiation in the presence of a conditioned medium.
Figure 2A tests the effect of prostaglandin PGD2 and its metabolites secreted by the senescent cells on the differentiation of myoblasts. Therefore, we inhibited the synthesis of PGD2 in senescent cells by treatment with AT-56, and then collected the conditioned medium. Conditioned medium collected from senescent C2C12 cells treated with vehicle (DMSO) served as a control for the experiment, whereas differentiation of C2C12 cells without any treatment serves as a positive control.
There is no explanation of how differentiation was quantified or how the fusion index was calculated.
The fusion index was calculated using a published myotube analyzer software (Noë et al., 2022). Appropriate info will be added to the materials and methods section in the revised manuscript.
