The transcription factor Slug represses p16Ink4a and regulates murine muscle stem cell aging.
ABSTRACT: Activation of the p16Ink4a-associated senescence pathway during aging breaks muscle homeostasis and causes degenerative muscle disease by irreversibly dampening satellite cell (SC) self-renewal capacity. Here, we report that the zinc-finger transcription factor Slug is highly expressed in quiescent SCs of mice and functions as a direct transcriptional repressor of p16Ink4a. Loss of Slug promotes derepression of p16Ink4a in SCs and accelerates the entry of SCs into a fully senescent state upon damage-induced stress. p16Ink4a depletion partially rescues defects in Slug-deficient SCs. Furthermore, reduced Slug expression is accompanied by p16Ink4a accumulation in aged SCs. Slug overexpression ameliorates aged muscle regeneration by enhancing SC self-renewal through active repression of p16Ink4a transcription. Our results identify a cell-autonomous mechanism underlying functional defects of SCs at advanced age. As p16Ink4a dysregulation is the chief cause for regenerative defects of human geriatric SCs, these findings highlight Slug as a potential therapeutic target for aging-associated degenerative muscle disease.
Project description:Sustainable muscle regeneration necessitates proper maintenance of the quiescence-reversible SCs pool. Activation of p16Ink4a-associated senescence pathway during aging breaks muscle homeostasis and causes degenerative muscle disease by irreversibly dampening satellite cell (SC) self-renewal capacity. We performed microarrays analysis to compare the genome-wide gene expression profiles of wild-type and Slug-deficient SCs and identified distinct classes of up-regulated genes upon deletion of Slug gene. Overall design: Quiescent SCs were collected by FACS sorting of CD45-Sca1-CD11b-CD31-CD34+Integrin-α7+ cell population in mononucleated cells prepared from hindlimb muslces of adult wildtype and Slug knockout mice, respectively. RNA was extracted for Affymetrix microarrays analysis.
Project description:With age, the epidermis becomes hypoplastic and hypoproliferative. Hypoproliferation due to aging has been associated with decreased stem cell (SC) self-renewal in multiple murine tissues. The fate of SC self-renewal divisions can be asymmetric (one SC, one committed progenitor) or symmetric (two SCs). Increased asymmetric SC self-renewal has been observed in inflammatory-mediated hyperproliferation, while increased symmetric SC self-renewal has been observed in cancers. We analyzed SC self-renewal divisions in aging human epidermis to better understand the role of SCs in the hypoproliferation of aging. In human subjects, neonatal to 78 years, there was an age-dependent decrease in epidermal basal layer divisions. The balance of SC self-renewal shifted toward symmetric SC self-renewal, with a decline in asymmetric SC self-renewal. Asymmetric SC divisions maintain epidermal stratification, and this decrease may contribute to the hypoplasia of aging skin. P53 decreases in multiple tissues with age, and p53 has been shown to promote asymmetric SC self-renewal. Fewer aged than adult ALDH+CD44+ keratinocyte SCs exhibited p53 expression and activity and Nutlin-3 (a p53 activator) returned p53 activity as well as asymmetric SC self-renewal divisions to adult levels. Nutlin-3 increased Notch signaling (NICD, Hes1) and DAPT inhibition of Notch activation prevented Nutlin-3 (p53)-induced asymmetric SC self-renewal divisions in aged keratinocytes. These studies indicate a role for p53 in the decreased asymmetric SC divisions with age and suggest that in aged keratinocytes, Notch is required for p53-induced asymmetric SC divisions.
Project description:In the neurogenic niches-the dentate gyrus of the hippocampus and the subventricular zone (SVZ) adjacent to lateral ventricles-stem cells continue to divide during adulthood, generating progenitor cells and new neurons, and to self-renew, thus maintaining the stem cell pool. During aging, the numbers of stem/progenitor cells in the neurogenic niches are reduced. The preservation of the neurogenic pool is committed to a number of antiproliferative genes, with the role of maintaining the quiescence of neural cells. The cyclin-dependent kinase inhibitor p16Ink4a, whose expression increases with age, controls the expansion of SVZ aging stem cells, since in mice its deficiency prevents the decline of neurogenesis in SVZ. No change of neurogenesis is however observed in the p16Ink4a-null dentate gyrus. Here, we hypothesized that p16Ink4a plays a role as a regulator of the self-renewal of the stem cell pool also in the dentate gyrus, and to test this possibility we stimulated the dentate gyrus neural cells of p16Ink4a-null aging mice with physical exercise, a powerful neurogenic activator. We observed that running highly induced the generation of new stem cells in the p16Ink4a-null dentate gyrus, forcing them to exit from quiescence. Stem cells, notably, are not induced to proliferate by running in wild-type (WT) mice. Moreover, p16Ink4a-null progenitor cells were increased by running significantly above the number observed in WT mice. The new stem and progenitor cells generated new neurons, and continued to actively proliferate in p16Ink4a-null mice longer than in the WT after cessation of exercise. Thus, p16Ink4a prevents aging dentate gyrus stem cells from being activated by exercise. Therefore, p16Ink4a may play a role in the maintenance of dentate gyrus stem cells after stimulus, by keeping a reserve of their self-renewal capacity during aging.
Project description:Skeletal muscle stem cells, i.e., satellite cells (SCs), are the essential source of new myonuclei for skeletal muscle regeneration following injury or chronic degenerative myopathies. Both SC number and regenerative capacity diminish during aging. However, molecular regulators that govern sizing of the initial SC pool are unknown. We demonstrate that fibroblast growth factor 6 (FGF6) is critical for SC pool scaling. Mice lacking FGF6 have reduced SCs of early postnatal origin and impaired regeneration. By contrast, increasing FGF6 during the early postnatal period is sufficient for SC expansion. Together, these data support that FGF6 is necessary and sufficient to modulate SC numbers during a critical postnatal period to establish the quiescent adult muscle stem cell pool. Our work highlights postnatal development as a time window receptive for scaling a somatic stem cell population via growth factor signaling, which might be relevant for designing new biomedical strategies to enhance tissue regeneration.
Project description:Skeletal muscle stem cells, or "satellite cells" (SCs), are required for the regeneration of damaged muscle tissue. Although SCs self-renew during regeneration, the mechanisms that govern SC re-entry into quiescence remain elusive. We show that FOXO3, a member of the forkhead family of transcription factors, is expressed in quiescent SCs (QSCs). Conditional deletion of Foxo3 in QSCs impairs self-renewal and increases the propensity of SCs to adopt a differentiated fate. Transcriptional analysis of SCs lacking FOXO3 revealed a downregulation of Notch signaling, a key regulator of SC quiescence. Conversely, overexpression of Notch intracellular domain (NICD) rescued the self-renewal deficit of FOXO3-deficient SCs. We show that FOXO3 regulates NOTCH1 and NOTCH3 receptor expression and that decreasing expression of NOTCH1 and NOTCH3 receptors phenocopies the effect of FOXO3 deficiency in SCs. We demonstrate that FOXO3, perhaps by activating Notch signaling, promotes the quiescent state during SC self-renewal in adult muscle regeneration.
Project description: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 PKC? in SC self-renewal and differentiation. We show that PKC? is expressed in SCs, and its active form is localized to the chromosomes, centrosomes, and midbody during mitosis. Lack of PKC? promotes SC symmetric self-renewal division by regulating Pard3 polarity protein localization, without affecting the overall proliferation rate. Genetic ablation of PKC? 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 PKC? resulted in a significant expansion of the quiescent SC pool. Finally, we show that lack of PKC? does not alter the inflammatory milieu after acute injury in muscle, suggesting that the enhanced self-renewal ability of SCs in PKC?-/- mice is not due to an alteration in the inflammatory milieu. Together, these results suggest that PKC? 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.
Project description:Human aging is associated with a decline in skeletal muscle (SkM) function and a reduction in the number and activity of satellite cells (SCs), the resident stem cells. To study the connection between SC aging and muscle impairment, we analyze the whole genome of single SC clones of the leg muscle vastus lateralis from healthy individuals of different ages (21-78 years). We find an accumulation rate of 13 somatic mutations per genome per year, consistent with proliferation of SCs in the healthy adult muscle. SkM-expressed genes are protected from mutations, but aging results in an increase in mutations in exons and promoters, targeting genes involved in SC activity and muscle function. In agreement with SC mutations affecting the whole tissue, we detect a missense mutation in a SC propagating to the muscle. Our results suggest somatic mutagenesis in SCs as a driving force in the age-related decline of SkM function.
Project description:<h4>Background</h4>Myopathic changes are commonly described in hypothyroid and hyperthyroid patients, including muscular atrophy and weakness. Satellite cells (SCs) play a major role in skeletal muscle maintenance and regeneration after injury. A mouse model of resistance to thyroid hormone-TR?1PV demonstrated impaired skeletal muscle regeneration after injury with significant reduction of SCs, suggesting that exhaustion of the SC pool contributes to the impaired regeneration. To test this hypothesis, SC activation and proliferation were analyzed in vivo in response to skeletal muscle injury and during aging.<h4>Methods</h4>SCs of TR?1PV male mice were analyzed four days after cardiotoxin-induced muscle injury, and they were compared to wild-type (WT) male animals. TR?-knockdown C2C12 myoblasts were injected into injured skeletal muscle, and four days after transplantation, the in vivo behavior was compared to control C2C12 myoblasts. Skeletal muscle regeneration was compared in younger and older TR?1PV and WT animals.<h4>Results</h4>The total number of SCs in skeletal muscle of TR?1PV mice was significantly lower than control, both before and shortly after muscle injury, with significant impairment of SC activation, consistent with SC pool exhaustion. TR?-knockdown myoblasts showed impaired in vivo proliferation and migration. TR?1PV mice had skeletal muscle loss and significant impairment in skeletal muscle regeneration with aging. This translated to a significant reduction of the SC pool with aging compared to WT mice.<h4>Conclusion</h4>TR? plays an important role in the maintenance of the SC pool. Impaired skeletal muscle regeneration in TR?1PV mice is associated with insufficient SC activation and proliferation, as well as the progressive loss of the SC pool with aging. Regulation of the SC pool and SC proliferation provides a therapeutic target to enhance skeletal muscle regeneration and possibly slow age-associated sarcopenia.
Project description:The remarkable regeneration capability of skeletal muscle depends on the coordinated proliferation and differentiation of satellite cells (SCs). The self-renewal of SCs is critical for long-term maintenance of muscle regeneration potential. Hypoxia profoundly affects the proliferation, differentiation, and self-renewal of cultured myoblasts. However, the physiological relevance of hypoxia and hypoxia signaling in SCs in vivo remains largely unknown. Here, we demonstrate that SCs are in an intrinsic hypoxic state in vivo and express hypoxia-inducible factor 2A (HIF2A). HIF2A promotes the stemness and long-term homeostatic maintenance of SCs by maintaining their quiescence, increasing their self-renewal, and blocking their myogenic differentiation. HIF2A stabilization in SCs cultured under normoxia augments their engraftment potential in regenerative muscle. Conversely, HIF2A ablation leads to the depletion of SCs and their consequent regenerative failure in the long-term. In contrast, transient pharmacological inhibition of HIF2A accelerates muscle regeneration by increasing SC proliferation and differentiation. Mechanistically, HIF2A induces the quiescence and self-renewal of SCs by binding the promoter of the Spry1 gene and activating Spry1 expression. These findings suggest that HIF2A is a pivotal mediator of hypoxia signaling in SCs and may be therapeutically targeted to improve muscle regeneration.
Project description:The function and number of muscle stem cells (satellite cells, SCs) decline with muscle aging. Although SCs are heterogeneous and different subpopulations have been identified, it remains unknown whether a specific subpopulation of muscle SCs selectively decreases during aging. Here, we find that the number of SCs expressing high level of transcription factor Pax7 (Pax7Hi ) is dramatically reduced in aged mice. Myofiber-secreted granulocyte colony-stimulating factor (G-CSF) regulates age-dependent loss of Pax7Hi cells, as the Pax7Hi SCs are replenished by exercise-induced G-CSF in aged mice. Mechanistically, we show that transcription of G-CSF (Csf3) gene in myofibers is regulated by MyoD in a metabolism-dependent manner. Furthermore, myofiber-secreted G-CSF acts as a metabolic niche factor required for establishing and maintaining the Pax7Hi SC subpopulation in adult and physiological aged mice by promoting the asymmetric division of Pax7Hi and Pax7Mi SCs. Together, our findings uncover that muscles provide a metabolic niche regulating Pax7 SC heterogeneity in mice.