PASK links cellular energy metabolism with a mitotic self-renewal network to establish differentiation competence

  1. Michael Xiao
  2. Chia-Hua Wu
  3. Graham Meek
  4. Brian Kelly
  5. Dara Buendia Castillo
  6. Lyndsay EA Young
  7. Sara Martire
  8. Sajina Dhungel
  9. Elizabeth McCauley
  10. Purbita Saha
  11. Altair L Dube
  12. Matthew S Gentry
  13. Laura A Banaszynski
  14. Ramon C Sun
  15. Chintan K Kikani

  • Curated by eLife

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    This study advances the understanding of metabolic regulation underpinning self-renewal of stem cells. The authors report that glutamine-dependent acetylation of the kinase PASK regulates its nuclear localization. Evidence is provided that nuclear PASK binds and disrupts Wdr5 association with the anaphase-promoting complex/cyclosome and is a trigger for the activation of myogenic programs in cultured cells. The study will be of interest to an audience in the areas of stem cells, regeneration and metabolic signalling.

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Abstract

Quiescent stem cells are activated in response to a mechanical or chemical injury to their tissue niche. Activated cells rapidly generate a heterogeneous progenitor population that regenerates the damaged tissues. While the transcriptional cadence that generates heterogeneity is known, the metabolic pathways influencing the transcriptional machinery to establish a heterogeneous progenitor population remains unclear. Here, we describe a novel pathway downstream of mitochondrial glutamine metabolism that confers stem cell heterogeneity and establishes differentiation competence by countering post-mitotic self-renewal machinery. We discovered that mitochondrial glutamine metabolism induces CBP/EP300-dependent acetylation of stem cell-specific kinase, PAS domain-containing kinase (PASK), resulting in its release from cytoplasmic granules and subsequent nuclear migration. In the nucleus, PASK catalytically outcompetes mitotic WDR5-anaphase-promoting complex/cyclosome (APC/C) interaction resulting in the loss of post-mitotic Pax7 expression and exit from self-renewal. In concordance with these findings, genetic or pharmacological inhibition of PASK or glutamine metabolism upregulated Pax7 expression, reduced stem cell heterogeneity , and blocked myogenesis in vitro and muscle regeneration in mice. These results explain a mechanism whereby stem cells co-opt the proliferative functions of glutamine metabolism to generate transcriptional heterogeneity and establish differentiation competence by countering the mitotic self-renewal network via nuclear PASK.

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  1. Version published to 10.7554/elife.81717 on eLife
  2. eLife

    Author Response

    Reviewer #2 (Public Review):

    In this paper, Xiao et al. suggest that PASK is a driver for stem cell differentiation by translocating from the cytosol to the nucleus. This phenomenon is dependent on the acetylation of PASK mediated by CBP/EP300, which is driven by glutamine metabolism. Furthermore, this study showed that PASK interferes/weakens the Wdr5-APC/C interaction, where PASK interacts with Wdr5, resulting in repression of Pax7, leading to stem cell differentiation.

    There exist huge interest in maintaining adult stem cells and ES cells in their pluripotent form and the work painstakingly perform several experiments to present that PASK is a good target to achieve that goal.

    However, the work on the paper relies mostly on data from C2C12 cells as adult muscle stem cell models, in vivo experimental data, and primary myoblasts from mice. Using these models makes the story contextual in muscle stem cells. Authors have not tried to extrapolate similar claims in other adult stem cell models. This severely restricts the claim to muscle stem cells even though PASK is required for the onset of embryonic and adult stem cell differentiation in general. Their work could be much strengthened if it is also tried on mesenchymal stem cells as these cells are also as metabolically active as muscle cells.

    We thank reviewers for their enthusiasm for our studies using PASKi. We have previously shown that PASKi prevented differentiation of 10T1/2 cells into adipogenic lineage (Kikani et al, Elife, 2016). We used stem cells from embryonic (ESC) and adult (MuSCs) origin to show broad application of PASKi in preserving self-renewal independent of stem cell origin. We believe that PASK function to be conversed across different stem cell paradigms; and our results in this manuscript would provide framework to further study PASK in other stem cell paradigms.

    Reviewer #3 (Public Review):

    This manuscript entitled "PASK relays metabolic signals to mitotic Wdr5-APC/C complex to drive exit from selfrenewal" by Xiao et al presents an interesting story on the role of PASK in the control of muscle stem cell fate by controlling the decision between self-renewal and differentiation. While the biochemistry presented is fairly compelling, the experiments revolving around the myogenic cells are lacking in quality and data.

    Major concerns:

    1. The isolation method used by this group to isolate muscle stem cells is inappropriate for the experiments used and may contribute to the misinterpretation of some of the results. It is simply a preplating method that results in a very heterogenous cell population in terms of cell type, comprised of numerous fibroblasts. While preplating can be used to isolate muscle stem cells and culture them as myoblasts, it takes days of growth and multiple rounds of passaging that are not used in this paper in order to get a more pure population of myogenic cells. This would also explain the high number of Pax7 negative cells in their primary myoblast experiments (~50% in some conditions) as they are most likely fibroblasts, which the authors could show by staining for fibroblast markers. The increase in Pax7 cells in certain conditions could also simply be due to the loss of contaminating cell types due to the treatment. Every single experiment that was performed on myoblasts must be redone using a more appropriate cell isolation method (i.e. FACS) or by culturing these isolated cells for a much longer period of time to eventually get a more pure cell population. As it stands, none of the data from the primary myoblast experiments are trustworthy.

    We agree – and thus, we have reproduced our results using two different methods of purifying MuSCs from mice, as indicated above. We took care to stain each isolation method with vimentin (a marker for fibroblasts) to ensure the purity of our preparation. Data are included in the Essential revisions section.

    1. The authors possess a genetic mouse model where PASK is knocked out. However, the mouse model is never described and the paper that is referenced also does not describe it. Please detail your mouse model.
    1. The majority of experiments are performed on C2C12 cells. While C2C12s are adequate for biochemistry and proof of concepts, when it comes to biological significance primary myoblasts should be used. While the authors try to explain this use by claiming that primary myoblasts undergo precocious differentiation that can be avoided by using an appropriate growth media (F10, 20% FBS, 1% P/S, 5ng/mL of bFGF).

    Kindly see the response for this comment in the Essential revision section.

    1. The authors possess a genetic mouse model, yet performed RNA-Seq on C2C12 myoblasts that were either untreated or treated with a PASK inhibitor. It would be much more informative and valuable to sequence the primary myoblasts from WT and PASK KO mice, thereby providing a more biologically relevant model.

    We used C2C12 for several reasons for initial transcriptome analysis using PASKi and validated the results from that analysis in primary myoblasts. (1) C2C12 cells are an excellent model for performing biochemical pathway characterization, including discovering new substrate targets for PASK, finding PASK interacting partners, and measuring the biochemical activity of PASK under various conditions. Thus, it would form the basis for a longer-term study of the signaling functions of PASK in one cell system (myoblasts), which can be validated and compared with the primary cell system. (2) PASKi treatment can acutely inhibit PASK catalytic activity without the genetic loss of its protein level. For many enzymatic proteins, catalytic inhibition could have a different biological effect compared with genetic loss of protein (Weiss et al.; Nat Chem Biol. 2007 Dec; 3(12): 739–744.). Thus, we chose the PASKi and C2C12 myoblasts system to study the kinase activitydependent effect on the myoblast transcriptome. However, throughout the manuscript, we used PASKi, PASK siRNA, and PASKKO primary cells to cross-validate all our data. We believe the conditional loss of PASK in MuSCs specific manner will be a great model to repeat the RNA-seq analysis in the future and compare the data obtained with PASKi in cultured myoblasts.

    1. The KO mouse model is rarely used and the cells isolated from it would be very useful in determining the biological role of PASK in muscle cells. The authors should isolate WT and KO cells and perform basic muscle functional experiments such as EDU incorporation for proliferation, and fusion index for differentiation to see whether the loss of PASK has an effect on these cells.

    We have published the characterization of myogenesis phenotype of PASKKO model in our previous manuscript (Kikani et al, 2016). Thus, we erred by not redoing those experiment in the previous version. We have now reproduced those results and markedly extended the chacterization of PASKKO cells in vitro, including BrdU incorporation, myogenesis, Pax7 heterogeneity, Myogenin expression and PASK subcellular distribution using WT cells. We have also characterized regeneration phenotype of PASKKO mice. We thank the reviewer for helping strengthen the biological context of our manuscript.

    1. The authors never look at quiescent muscle stem cells and early activated muscle stem cells in terms of PASK protein expression and dynamics. The authors should isolate EDL myofibers and stain for PASK and PAX7 at 0, 24, 48, and 72-hour post isolation. This would allow the authors to quantify the changes in PASK expression and cell localization, as well as confirm the number of muscle stem cells in WT and KO mice, during quiescence and during the process of muscle stem cell activation, proliferation, and differentiation in a near in vivo context.

    As described in Figure 1-figure supplement 2A, PASK is not expressed in quiescent MuSCs. Therefore, we do not anticipate a functional role of PASK in initial activation of QSC. We do not propose that PASK plays a role in the maintenance of the QSC state or the exit and initial activation of MuSCs following muscle injury. PASK is transcriptionally activated in proliferating myoblasts during regeneration (Kikani et al, elife 2016) and upon isolation of MuSCs (Figure S1D). Therefore, we specifically focus on studying the biochemical functional role of PASK signaling in activated (proliferating) myoblasts isolated from mice or during early regeneration. We have ongoing studies examining the precise temporal kinetics of PASK transcription regulation in Pax7+ MuSCs as they are activated, and to identify its upstream transcriptional regulators. However, we respectfully suggest that these avenues are outside of the purview of this current manuscript that specifically explores the metabolic pathway that establishes progenitor population from activated myoblasts.

    1. Contrary to their claim, MyoD is not a stemness/self-renewal gene.

    We agree, and have corrected the text.

    1. The authors state that PASK is necessary for exit from self-renewal and establishment of a progenitor population, but this is a vast overstatement. In the genetic KO mouse model, the mice are able to regenerate their muscle after injury, therefore PASK cannot be a necessary protein for the formation of progenitor cells.

    During the muscle regeneration, we observed a significant inhibition of the early regenerative response in PASKKO mice, marked by severely reduced levels of eMHC. Concomittantly, we observed increased numbers of Pax7+ MuSCs at Day 5 of regeneration compared with WT muscles. We have extensively shown requirement of PASK for myogenin induction in vitro and in vivo (Kikani et al, 2016, Kikani et al, 2019). Based on these evidence, we propose that PASK is necessary for the exit from Pax7+ self-renewing stem cells and generation of Myog+ committed progenitor populations.

    1. In numerous figure panels, the y-axis represents the # of cells, rather than a percentage or ratio. This is uninformative as the number of cells will never be the same between conditions and experiments. These panels need to be replaced with a more appropriate y-axis.

    We have updated the axes to % cells where appropriate.

  3. eLife

    eLife assessment

    This study advances the understanding of metabolic regulation underpinning self-renewal of stem cells. The authors report that glutamine-dependent acetylation of the kinase PASK regulates its nuclear localization. Evidence is provided that nuclear PASK binds and disrupts Wdr5 association with the anaphase-promoting complex/cyclosome and is a trigger for the activation of myogenic programs in cultured cells. The study will be of interest to an audience in the areas of stem cells, regeneration and metabolic signalling.

  4. eLife

    Reviewer #1 (Public Review):

    The study provides mechanistic insight into molecular events occurring at the onset of differentiation mediated by the kinase PASK. Specifically, the work focuses on the multiple steps that converge on post-translational modifications of PASK and its translocation to the nucleus during myogenesis. The authors present evidence that glutamine-fueled, CPB/EP300-mediated acetylation of PASK is required for its nuclear translocation. This allows (nuclear) PASK to interact with Wdr5 and consequently disrupt its association with the anaphase-promoting complex/cyclosome and inhibit Pax7 transcription, marking the onset of muscle differentiation. The conclusions are supported by an analysis of the effects of glutamine modulation on differentiation and maintenance of stemness in primary muscle stem cells; PASK localization in myoblasts and primary muscle stem cells as well as detailed biochemistry with modified forms of PASK to interrogate molecular interactions. C2C12 myoblast cells and primary muscle stem cells are cellular systems employed in the study with observations confirmed in cells derived from mice with genetic ablation of PASK. The study provides molecular detail on events linking glutamine metabolism to the transcriptional control of lineage differentiation, through the regulation of PASK. The analysis of these events in other systems would be of value to understanding their broader applicability.

  5. eLife

    Reviewer #2 (Public Review):

    In this paper, Xiao et al. suggest that PASK is a driver for stem cell differentiation by translocating from the cytosol to the nucleus. This phenomenon is dependent on the acetylation of PASK mediated by CBP/EP300, which is driven by glutamine metabolism. Furthermore, this study showed that PASK interferes/weakens the Wdr5-APC/C interaction, where PASK interacts with Wdr5, resulting in repression of Pax7, leading to stem cell differentiation.

    There exist huge interest in maintaining adult stem cells and ES cells in their pluripotent form and the work painstakingly perform several experiments to present that PASK is a good target to achieve that goal.

    However, the work on the paper relies mostly on data from C2C12 cells as adult muscle stem cell models, in vivo experimental data, and primary myoblasts from mice. Using these models makes the story contextual in muscle stem cells. Authors have not tried to extrapolate similar claims in other adult stem cell models. This severely restricts the claim to muscle stem cells even though PASK is required for the onset of embryonic and adult stem cell differentiation in general. Their work could be much strengthened if it is also tried on mesenchymal stem cells as these cells are also as metabolically active as muscle cells.

  6. eLife

    Reviewer #3 (Public Review):

    This manuscript entitled "PASK relays metabolic signals to mitotic Wdr5-APC/C complex to drive exit from self-renewal" by Xiao et al presents an interesting story on the role of PASK in the control of muscle stem cell fate by controlling the decision between self-renewal and differentiation. While the biochemistry presented is fairly compelling, the experiments revolving around the myogenic cells are lacking in quality and data.

    Major concerns:

    1. The isolation method used by this group to isolate muscle stem cells is inappropriate for the experiments used and may contribute to the misinterpretation of some of the results. It is simply a preplating method that results in a very heterogenous cell population in terms of cell type, comprised of numerous fibroblasts. While preplating can be used to isolate muscle stem cells and culture them as myoblasts, it takes days of growth and multiple rounds of passaging that are not used in this paper in order to get a more pure population of myogenic cells. This would also explain the high number of Pax7 negative cells in their primary myoblast experiments (~50% in some conditions) as they are most likely fibroblasts, which the authors could show by staining for fibroblast markers. The increase in Pax7 cells in certain conditions could also simply be due to the loss of contaminating cell types due to the treatment. Every single experiment that was performed on myoblasts must be redone using a more appropriate cell isolation method (i.e. FACS) or by culturing these isolated cells for a much longer period of time to eventually get a more pure cell population. As it stands, none of the data from the primary myoblast experiments are trustworthy.
    2. The authors possess a genetic mouse model where PASK is knocked out. However, the mouse model is never described and the paper that is referenced also does not describe it. Please detail your mouse model.
    3. The majority of experiments are performed on C2C12 cells. While C2C12s are adequate for biochemistry and proof of concepts, when it comes to biological significance primary myoblasts should be used. While the authors try to explain this use by claiming that primary myoblasts undergo precocious differentiation that can be avoided by using an appropriate growth media (F10, 20% FBS, 1% P/S, 5ng/mL of bFGF).
    4. The authors possess a genetic mouse model, yet performed RNA-Seq on C2C12 myoblasts that were either untreated or treated with a PASK inhibitor. It would be much more informative and valuable to sequence the primary myoblasts from WT and PASK KO mice, thereby providing a more biologically relevant model.
    5. The KO mouse model is rarely used and the cells isolated from it would be very useful in determining the biological role of PASK in muscle cells. The authors should isolate WT and KO cells and perform basic muscle functional experiments such as EDU incorporation for proliferation, and fusion index for differentiation to see whether the loss of PASK has an effect on these cells.
    6. The authors never look at quiescent muscle stem cells and early activated muscle stem cells in terms of PASK protein expression and dynamics. The authors should isolate EDL myofibers and stain for PASK and PAX7 at 0, 24, 48, and 72-hour post isolation. This would allow the authors to quantify the changes in PASK expression and cell localization, as well as confirm the number of muscle stem cells in WT and KO mice, during quiescence and during the process of muscle stem cell activation, proliferation, and differentiation in a near in vivo context.
    7. Contrary to their claim, MyoD is not a stemness/self-renewal gene.
    8. The authors state that PASK is necessary for exit from self-renewal and establishment of a progenitor population but this is a vast overstatement. In the genetic KO mouse model, the mice are able to regenerate their muscle after injury, therefore PASK cannot be a necessary protein for the formation of progenitor cells.
    9. In numerous figure panels, the y-axis represents the # of cells, rather than a percentage or ratio. This is uninformative as the number of cells will never be the same between conditions and experiments. These panels need to be replaced with a more appropriate y-axis.

  7. Version published to 10.1101/2022.03.24.485713v1 on bioRxiv