Targeting PKCθ Promotes Satellite Cell Self-Renewal

Skeletal muscle regeneration following injury depends on the ability of satellite cells (SCs) to proliferate, self-renew, and eventually differentiate. The factors that regulate the process of self-renewal are poorly understood. In this study we examined the role of PKCθ in SC self-renewal and differentiation. We show that PKCθ is expressed in SCs, and its active form is localized to the chromosomes, centrosomes, and midbody during mitosis. Lack of PKCθ promotes SC symmetric self-renewal division by regulating Pard3 polarity protein localization, without affecting the overall proliferation rate. Genetic ablation of PKCθ or its pharmacological inhibition in vivo did not affect SC number in healthy muscle. By contrast, after induction of muscle injury, lack or inhibition of PKCθ resulted in a significant expansion of the quiescent SC pool. Finally, we show that lack of PKCθ does not alter the inflammatory milieu after acute injury in muscle, suggesting that the enhanced self-renewal ability of SCs in PKCθ-/- mice is not due to an alteration in the inflammatory milieu. Together, these results suggest that PKCθ plays an important role in SC self-renewal by stimulating their expansion through symmetric division, and it may represent a promising target to manipulate satellite cell self-renewal in pathological conditions.

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MLL1 is required for PAX7 expression and satellite cell self-renewal in mice

Nature Communications ◽

10.1038/s41467-019-12086-9 ◽

2019 ◽

Vol 10 (1) ◽

Author(s):

Gregory C. Addicks ◽

Caroline E. Brun ◽

Marie-Claude Sincennes ◽

John Saber ◽

Christopher J. Porter ◽

...

Keyword(s):

Satellite Cell ◽

Satellite Cells ◽

Transcriptional Activation ◽

Cell Activation ◽

Target Genes ◽

Cell Function ◽

Skeletal Muscle Regeneration ◽

Differentiation Potential ◽

Self Renewal

Abstract PAX7 is a paired-homeobox transcription factor that specifies the myogenic identity of muscle stem cells and acts as a nodal factor by stimulating proliferation while inhibiting differentiation. We previously found that PAX7 recruits the H3K4 methyltransferases MLL1/2 to epigenetically activate target genes. Here we report that in the absence of Mll1, myoblasts exhibit reduced H3K4me3 at both Pax7 and Myf5 promoters and reduced Pax7 and Myf5 expression. Mll1-deficient myoblasts fail to proliferate but retain their differentiation potential, while deletion of Mll2 had no discernable effect. Re-expression of PAX7 in committed Mll1 cKO myoblasts restored H3K4me3 enrichment at the Myf5 promoter and Myf5 expression. Deletion of Mll1 in satellite cells reduced satellite cell proliferation and self-renewal, and significantly impaired skeletal muscle regeneration. Pax7 expression was unaffected in quiescent satellite cells but was markedly downregulated following satellite cell activation. Therefore, MLL1 is required for PAX7 expression and satellite cell function in vivo. Furthermore, PAX7, but not MLL1, is required for Myf5 transcriptional activation in committed myoblasts.

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PEDF-derived peptide promotes skeletal muscle regeneration through its mitogenic effect on muscle progenitor cells

AJP Cell Physiology ◽

10.1152/ajpcell.00344.2014 ◽

2015 ◽

Vol 309 (3) ◽

pp. C159-C168 ◽

Cited By ~ 18

Author(s):

Tsung-Chuan Ho ◽

Yi-Pin Chiang ◽

Chih-Kuang Chuang ◽

Show-Li Chen ◽

Jui-Wen Hsieh ◽

...

Keyword(s):

Skeletal Muscle ◽

Cell Proliferation ◽

Cyclin D1 ◽

Satellite Cell ◽

Muscle Regeneration ◽

Muscle Fibers ◽

Skeletal Muscle Regeneration ◽

Satellite Cell Proliferation

In response injury, intrinsic repair mechanisms are activated in skeletal muscle to replace the damaged muscle fibers with new muscle fibers. The regeneration process starts with the proliferation of satellite cells to give rise to myoblasts, which subsequently differentiate terminally into myofibers. Here, we investigated the promotion effect of pigment epithelial-derived factor (PEDF) on muscle regeneration. We report that PEDF and a synthetic PEDF-derived short peptide (PSP; residues Ser93-Leu112) induce satellite cell proliferation in vitro and promote muscle regeneration in vivo. Extensively, soleus muscle necrosis was induced in rats by bupivacaine, and an injectable alginate gel was used to release the PSP in the injured muscle. PSP delivery was found to stimulate satellite cell proliferation in damaged muscle and enhance the growth of regenerating myofibers, with complete regeneration of normal muscle mass by 2 wk. In cell culture, PEDF/PSP stimulated C2C12 myoblast proliferation, together with a rise in cyclin D1 expression. PEDF induced the phosphorylation of ERK1/2, Akt, and STAT3 in C2C12 myoblasts. Blocking the activity of ERK, Akt, or STAT3 with pharmacological inhibitors attenuated the effects of PEDF/PSP on the induction of C2C12 cell proliferation and cyclin D1 expression. Moreover, 5-bromo-2′-deoxyuridine pulse-labeling demonstrated that PEDF/PSP stimulated primary rat satellite cell proliferation in myofibers in vitro. In summary, we report for the first time that PSP is capable of promoting the regeneration of skeletal muscle. The signaling mechanism involves the ERK, AKT, and STAT3 pathways. These results show the potential utility of this PEDF peptide for muscle regeneration.

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Myostatin negatively regulates satellite cell activation and self-renewal

The Journal of Cell Biology ◽

10.1083/jcb.200207056 ◽

2003 ◽

Vol 162 (6) ◽

pp. 1135-1147 ◽

Cited By ~ 483

Author(s):

Seumas McCroskery ◽

Mark Thomas ◽

Linda Maxwell ◽

Mridula Sharma ◽

Ravi Kambadur

Keyword(s):

Satellite Cell ◽

Satellite Cells ◽

Cell Activation ◽

Unit Length ◽

Immunohistochemical Analysis ◽

Cell Number ◽

Negative Regulator ◽

Self Renewal ◽

Satellite Cell Activation

Satellite cells are quiescent muscle stem cells that promote postnatal muscle growth and repair. Here we show that myostatin, a TGF-β member, signals satellite cell quiescence and also negatively regulates satellite cell self-renewal. BrdU labeling in vivo revealed that, among the Myostatin-deficient satellite cells, higher numbers of satellite cells are activated as compared with wild type. In contrast, addition of Myostatin to myofiber explant cultures inhibits satellite cell activation. Cell cycle analysis confirms that Myostatin up-regulated p21, a Cdk inhibitor, and decreased the levels and activity of Cdk2 protein in satellite cells. Hence, Myostatin negatively regulates the G1 to S progression and thus maintains the quiescent status of satellite cells. Immunohistochemical analysis with CD34 antibodies indicates that there is an increased number of satellite cells per unit length of freshly isolated Mstn−/− muscle fibers. Determination of proliferation rate suggests that this elevation in satellite cell number could be due to increased self-renewal and delayed expression of the differentiation gene (myogenin) in Mstn−/− adult myoblasts. Taken together, these results suggest that Myostatin is a potent negative regulator of satellite cell activation and thus signals the quiescence of satellite cells.

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Heterogeneity of satellite cells implicates DELTA1/NOTCH2 signaling in self-renewal

10.1101/824359 ◽

2019 ◽

Author(s):

Valeria Yartseva ◽

Leonard D. Goldstein ◽

Julia Rodman ◽

Lance Kates ◽

Mark Z. Chen ◽

...

Keyword(s):

Cell Fate ◽

Satellite Cell ◽

Satellite Cells ◽

Receptor Expression ◽

Self Renewal ◽

Cell Fate Decisions ◽

Muscle Homeostasis ◽

Early Regeneration ◽

Time Point

SUMMARYHow satellite cells and their progenitors balance differentiation and self-renewal to achieve sustainable tissue regeneration is not well understood. A major roadblock to understanding satellite cell fate decisions has been the difficulty to study this process in vivo. By visualizing expression dynamics of myogenic transcription factors during early regeneration in vivo, we identified the time point at which cells undergo decisions to differentiate or self-renew. Single-cell RNA sequencing revealed heterogeneity of satellite cells during both muscle homeostasis and regeneration, including a subpopulation enriched in Notch2 receptor expression. Furthermore, we reveal that differentiating cells express the Dll1 ligand. Using antagonistic antibodies we demonstrate that the DLL1 and NOTCH2 signaling pair is required for satellite cell self-renewal. Thus, differentiating cells provide the self-renewing signal during regeneration, enabling proportional regeneration in response to injury while maintaining the satellite cell pool. These findings have implications for therapeutic control of muscle regeneration.

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GPR84, a Proinflammatory Receptor, Sustains Wnt/β-Catenin Signaling In Leukemic Stem Cells For Maintenance Of MLL-AF9-Induced Leukemogenesis

Blood ◽

10.1182/blood.v122.21.3781.3781 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3781-3781

Author(s):

Philipp A Dietrich ◽

Murray D Norris ◽

Jenny Yingzi Wang

Keyword(s):

Stem Cells ◽

Conflicts Of Interest ◽

Active Form ◽

Leukemic Stem Cells ◽

Poor Survival ◽

Functional Link ◽

Self Renewal ◽

Hematopoietic Stem

Abstract Inappropriate activation of Wnt/β-catenin signaling confers hematopoietic progenitors the property of self-renewal that promotes malignant transformation in MLL-rearranged acute myeloid leukemia (AML). However, it has been noted that activation of β-catenin is observed in tumors without clear mutations in the major components of the pathway or increase in Wnt signaling. This suggests that other developmental signaling pathways may be capable of inducing activation or downstream signaling of β-catenin. Recently, a number of G protein-coupled receptors (GPCRs) have been shown to activate β-catenin signaling to recruit the key downstream components of the canonical Wnt pathway in distinct cell types, including stem cells. GPCRs, the largest family of cell-surface molecules involved in signal transmission, have emerged as crucial players in tumor growth and metastasis, and represent one of the most important drug targets in pharmaceutical development. Given the close functional link with activation of β-catenin signaling, a GPCR signaling pathway may act as the upstream regulator of β-catenin signaling in the establishment of leukemic stem cells (LSC). In this study, our microarray analysis comparing genes differentially expressed between LSC and normal hematopoietic stem cells (HSC) identified GPR84, a proinflammatory GPCR, as a potential LSC-specific candidate target. An analysis of the comprehensive patient outcome database (Oncogenomics – maintained by the National Cancer Institute) showed that high levels of GPR84 were significantly associated with poor survival in patients with leukemia (P=0.0048), implying its potential clinical relevance in predicting disease prognosis. Western blot and flow cytometric analyses confirmed the microarray results and revealed a positive correlation between GPR84 and β-catenin expression. We previously demonstrated that β-catenin was highly expressed in HSC transformed by MLL-AF9 (HSC-MLLAF9) and had lower expression in HSC transduced with leukemic oncogenes Hoxa9/Meis1 (HSC-Hoxa9/Meis1), while increased β-catenin expression was correlated with a poor survival rate in vivo. Herein, our results showed that forced expression of GPR84 induced a robust upregulation of β-catenin in HSC-Hoxa9/Meis1. Conversely, shRNA-mediated ablation of GPR84 in HSC-MLLAF9 led to highly significant downregulation of both GPR84 (P=0.0003) and β-catenin (P=0.0008). Further in vitro functional studies showed that GPR84 knockdown significantly reduced HSC-MLL-AF9 colony forming units (P=0.0006), and induced a marked reduction of cells in S-phase (P=0.0017). This deficient phenotype could be rescued by expression of a constitutively active form of β-catenin. Importantly, subsequent in vivo survival studies using leukemia transplantation mouse models showed that GPR84 knockdown significantly reduced LSC frequency and severely impaired maintenance (P<0.0001; 11 mice per cohort) of HSC-MLL-AF9 induced leukemia, a highly aggressive and drug-resistant subtype of AML. The defect in disease phenotype resulted from inhibited expression of both GPR84 and β-catenin. Furthermore, forced overexpression of GPR84 alone was not sufficient for leukemic transformation of HSC but conferred a growth advantage in vivo to HSC-Hoxa9/Meis1 cells and significantly accelerated the onset of Hoxa9/Meis1-induced AML (P=0.0039), establishing a completely malignant phenotype similar to HSC-MLL-AF9 in vivo (P=0.9986). These data support an oncogenic role of GPR84 in MLL-AF9-induced leukemogenesis. In conclusion, our studies have identified a novel β-catenin regulator that contributes to leukemia maintenance by sustaining aberrant activation of a stem cell self-renewal pathway in LSC, and drugs targeting GPR84 may represent a novel and promising strategy for improving the therapy and outcome of AML patients. Disclosures: No relevant conflicts of interest to declare.

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1512869112 ◽

2015 ◽

Vol 112 (38) ◽

pp. E5246-E5252 ◽

Cited By ~ 36

Author(s):

Sarah A. Dick ◽

Natasha C. Chang ◽

Nicolas A. Dumont ◽

Ryan A. V. Bell ◽

Charis Putinski ◽

...

Keyword(s):

Skeletal Muscle ◽

Satellite Cells ◽

Posttranslational Modifications ◽

Caspase 3 ◽

Myogenic Differentiation ◽

Skeletal Muscle Regeneration ◽

Stem Cell Population ◽

Myoblast Differentiation ◽

Self Renewal

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.

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Vitamin D Promotes Skeletal Muscle Regeneration and Mitochondrial Health

Frontiers in Physiology ◽

10.3389/fphys.2021.660498 ◽

2021 ◽

Vol 12 ◽

Author(s):

Christine M. Latham ◽

Camille R. Brightwell ◽

Alexander R. Keeble ◽

Brooke D. Munson ◽

Nicholas T. Thomas ◽

...

Keyword(s):

Skeletal Muscle ◽

Vitamin D ◽

Muscle Damage ◽

Muscle Regeneration ◽

Muscle Injury ◽

Time Course ◽

Active Form ◽

Skeletal Muscle Regeneration ◽

Optimal Timing

Vitamin D is an essential nutrient for the maintenance of skeletal muscle and bone health. The vitamin D receptor (VDR) is present in muscle, as is CYP27B1, the enzyme that hydroxylates 25(OH)D to its active form, 1,25(OH)D. Furthermore, mounting evidence suggests that vitamin D may play an important role during muscle damage and regeneration. Muscle damage is characterized by compromised muscle fiber architecture, disruption of contractile protein integrity, and mitochondrial dysfunction. Muscle regeneration is a complex process that involves restoration of mitochondrial function and activation of satellite cells (SC), the resident skeletal muscle stem cells. VDR expression is strongly upregulated following injury, particularly in central nuclei and SCs in animal models of muscle injury. Mechanistic studies provide some insight into the possible role of vitamin D activity in injured muscle. In vitro and in vivo rodent studies show that vitamin D mitigates reactive oxygen species (ROS) production, augments antioxidant capacity, and prevents oxidative stress, a common antagonist in muscle damage. Additionally, VDR knockdown results in decreased mitochondrial oxidative capacity and ATP production, suggesting that vitamin D is crucial for mitochondrial oxidative phosphorylation capacity; an important driver of muscle regeneration. Vitamin D regulation of mitochondrial health may also have implications for SC activity and self-renewal capacity, which could further affect muscle regeneration. However, the optimal timing, form and dose of vitamin D, as well as the mechanism by which vitamin D contributes to maintenance and restoration of muscle strength following injury, have not been determined. More research is needed to determine mechanistic action of 1,25(OH)D on mitochondria and SCs, as well as how this action manifests following muscle injury in vivo. Moreover, standardization in vitamin D sufficiency cut-points, time-course study of the efficacy of vitamin D administration, and comparison of multiple analogs of vitamin D are necessary to elucidate the potential of vitamin D as a significant contributor to muscle regeneration following injury. Here we will review the contribution of vitamin D to skeletal muscle regeneration following injury.

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Transforming Growth Factor-β1-Mediated Neuroprotection against Excitotoxic Injury in Vivo

Journal of Cerebral Blood Flow & Metabolism ◽

10.1097/01.wcb.0000090080.64176.44 ◽

2003 ◽

Vol 23 (10) ◽

pp. 1174-1182 ◽

Cited By ~ 85

Author(s):

Delphine Boche ◽

Colm Cunningham ◽

Jack Gauldie ◽

V Hugh Perry

Keyword(s):

Growth Factor ◽

Bacterial Infection ◽

Transforming Growth Factor Beta ◽

Transforming Growth Factor ◽

Active Form ◽

Acute Injury ◽

Cresyl Violet ◽

Excitotoxic Lesion ◽

Tgf Β1

Ischemic preconditioning is a phenomenon that describes how a sublethal ischemic insult can induce tolerance to subsequent ischemia. This phenomenon has been observed after focal or global ischemia in different animal models. However, the hypothesis that bacterial infection might lead to neuronal tolerance to injury has not been investigated. To mimic cerebral bacterial infection, we injected bacterial lipopolysaccharide (LPS) in the right dorsal hippocampus, followed 24 hours later by an excitotoxic lesion using kainic acid in the mouse model. Quantification of lesion size after cresyl violet counterstaining revealed that LPS pretreatment afforded neuroprotection to CA3 neurons against KA challenge. To investigate the events underlying this protection, we studied the cytokine profile induced after LPS injection. Interleukin (IL)-1β and transforming growth factor beta 1 (TGF-β1) were the main cytokines expressed at 24 hours after LPS injection. Because IL-1β has been described as deleterious in acute injury, we decided to investigate the function of TGF-β1. An adenovirus expressing a constitutively active form of TGF-β1 was injected intracerebrally 1 week before the induction of excitotoxic lesion, and neuronal protection was observed. To confirm the neuroprotective role of TGF-β1, the TGF-β1 adenovirus was replaced by recombinant human TGF-β1 protein and total neuroprotection was observed. Furthermore, the antibody-mediated blocking of TGF-β1 action prevented the protective effect of pretreatment with LPS. We have demonstrated in vivo that the cerebral tolerance phenomenon induced by LPS pretreatment is mediated by TGF-β1 cytokine.

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VDR and CYP27B1 are expressed in C2C12 cells and regenerating skeletal muscle: potential role in suppression of myoblast proliferation

AJP Cell Physiology ◽

10.1152/ajpcell.00014.2012 ◽

2012 ◽

Vol 303 (4) ◽

pp. C396-C405 ◽

Cited By ~ 96

Author(s):

Ratchakrit Srikuea ◽

Xiping Zhang ◽

Ok-Kyong Park-Sarge ◽

Karyn A. Esser

Keyword(s):

Skeletal Muscle ◽

Vitamin D ◽

Cell Proliferation ◽

Potential Role ◽

Active Form ◽

C2c12 Cells ◽

Skeletal Muscle Regeneration ◽

Western Blots ◽

C2c12 Myoblasts

1α,25(OH)2D3, the active form of vitamin D3, has been reported to regulate the cell biology of skeletal muscle. However, there has been some controversy about the expression of the vitamin D receptor (VDR) and thus the potential role of vitamin D3 in skeletal muscle. In this study, we isolated and sequenced the full-length Vdr and Cyp27b1 transcripts in C2C12 myoblasts and myotubes. Western blots and immunocytochemistry confirmed protein expression in both myoblasts and myotubes clearly demonstrating that C2C12 cells express VDR and CYP27B1. To determine the vitamin D3 action, we found that C2C12 myoblasts treated with either 1α,25(OH)2D3 or 25(OH)D3 inhibited cell proliferation and this was associated with increased Vdr expression. The observation that treatment of C2C12 myoblasts with the inactive form of vitamin D3, [25(OH)D3], inhibited proliferation suggested that CYP27B1 was functionally active. We used small interfering RNA to knock down Cyp27b1 in myoblasts, and cells were treated with 25(OH)D3. The growth-suppressive effects of 25(OH)D3 were abolished, suggesting that CYP27B1 in myoblasts is necessary for the ability of 25(OH)D3 to affect cell proliferation. Finally, we analyzed expression of VDR and CYP27B1 in regenerating skeletal muscle in vivo. We found that expression of VDR and CYP27B1 increased significantly at day 7 of regeneration, and these results confirm the expression of Vdr and Cyp27b1 in vivo and suggest a potential role for vitamin D3 in skeletal muscle regeneration following injury.

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