Aged Stem Cells Reprogram Their Daily Rhythmic Functions to Adapt to Stress.

Normal homeostatic functions of adult stem cells have rhythmic daily oscillations that are believed to become arrhythmic during aging. Unexpectedly, we find that aged mice remain behaviorally circadian and that their epidermal and muscle stem cells retain a robustly rhythmic core circadian machinery. However, the oscillating transcriptome is extensively reprogrammed in aged stem cells, switching from genes involved in homeostasis to those involved in tissue-specific stresses, such as DNA damage or inefficient autophagy. Importantly, deletion of circadian clock components did not reproduce the hallmarks of this reprogramming, underscoring that rewiring, rather than arrhythmia, is associated with physiological aging. While age-associated rewiring of the oscillatory diurnal transcriptome is not recapitulated by a high-fat diet in young adult mice, it is significantly prevented by long-term caloric restriction in aged mice. Thus, stem cells rewire their diurnal timed functions to adapt to metabolic cues and to tissue-specific age-related traits.

Geriatric muscle stem cells switch reversible quiescence into senescence.

Regeneration of skeletal muscle depends on a population of adult stem cells (satellite cells) that remain quiescent throughout life. Satellite cell regenerative functions decline with ageing. Here we report that geriatric satellite cells are incapable of maintaining their normal quiescent state in muscle homeostatic conditions, and that this irreversibly affects their intrinsic regenerative and self-renewal capacities. In geriatric mice, resting satellite cells lose reversible quiescence by switching to an irreversible pre-senescence state, caused by derepression of p16(INK4a) (also called Cdkn2a). On injury, these cells fail to activate and expand, undergoing accelerated entry into a full senescence state (geroconversion), even in a youthful environment. p16(INK4a) silencing in geriatric satellite cells restores quiescence and muscle regenerative functions. Our results demonstrate that maintenance of quiescence in adult life depends on the active repression of senescence pathways. As p16(INK4a) is dysregulated in human geriatric satellite cells, these findings provide the basis for stem-cell rejuvenation in sarcopenic muscles.

Monoclonal antibodies against receptor-induced binding sites detect cell-bound plasminogen in blood.

Binding of Glu-plasminogen (the native, circulating form of the zymogen) to cells results in enhancement of its activation. Cell-associated plasmin proteolytic activity is a key component of physiologic and pathologic processes requiring extracellular matrix degradation. Recently, we developed antiplasminogen mAbs that recognize receptor-induced binding sites (RIBS) in Glu-plasminogen and, therefore, preferentially react with cell-associated Glu-plasminogen in the presence of soluble Glu-plasminogen. Here we have used FACS with a representative antiplasminogen receptor-induced binding site mAb, mAb49, to examine whether plasminogen associates with peripheral blood cells in blood. Plasminogen binding to neutrophils, monocytes, B-lymphocytes, T-lymphocytes, and platelets was clearly detected. Treatment of whole blood with lipopolysaccharide or 12-0 tetradecanoylphorbol-13-acetate up-regulated plasminogen binding to neutrophils and in vivo treatment with all-trans retinoic acid decreased plasminogen binding to acute promyelocytic leukemia blasts. Our results demonstrate that mAb49 can be used to monitor cell-bound plasminogen in blood under both normal and pathologic conditions.

Interleukin-6 is an essential regulator of satellite cell-mediated skeletal muscle hypertrophy.

Skeletal muscles adapt to increasing workload by augmenting their fiber size, through mechanisms that are poorly understood. This study identifies the cytokine interleukin-6 (IL-6) as an essential regulator of satellite cell (muscle stem cell)-mediated hypertrophic muscle growth. IL-6 is locally and transiently produced by growing myofibers and associated satellite cells, and genetic loss of IL-6 blunted muscle hypertrophy in vivo. IL-6 deficiency abrogated satellite cell proliferation and myonuclear accretion in the preexisting myofiber by impairing STAT3 activation and expression of its target gene cyclin D1. The growth defect was indeed muscle cell intrinsic, since IL-6 loss also affected satellite cell behavior in vitro, in a STAT3-dependent manner. Myotube-produced IL-6 further stimulated cell proliferation in a paracrine fashion. These findings unveil a role for IL-6 in hypertrophic muscle growth and provide mechanistic evidence for the contribution of satellite cells to this process.

Monoclonal antibodies detect receptor-induced binding sites in Glu-plasminogen.

When Glu-plasminogen binds to cells, its activation to plasmin is markedly enhanced compared with the reaction in solution, suggesting that Glu-plasminogen on cell surfaces adopts a conformation distinct from that in solution. However, direct evidence for such conformational changes has not been obtained. Therefore, we developed anti-plasminogen mAbs to test the hypothesis that Glu-plasminogen undergoes conformational changes on its interaction with cells. Six anti-plasminogen mAbs (recognizing 3 distinct epitopes) that preferentially recognized receptor-induced binding sites (RIBS) in Glu-plasminogen were obtained. The mAbs also preferentially recognized Glu-plasminogen bound to the C-terminal peptide of the plasminogen receptor, Plg-R(KT), and to fibrin, plasmin-treated fibrinogen, and Matrigel. We used trypsin proteolysis, immunoaffinity chromatography, and tandem mass spectrometry and identified Glu-plasminogen sequences containing epitopes recognized by the anti-plasminogen-RIBS mAbs: a linear epitope within a domain linking kringles 1 and 2; a nonlinear epitope contained within the kringle 5 domain and the latent protease domain; and a nonlinear epitope contained within the N-terminal peptide of Glu-plasminogen and the latent protease domain. Our results identify neoepitopes latent in soluble Glu-plasminogen that become available when Glu-plasminogen binds to cells and demonstrate that binding of Glu-plasminogen to cells induces a conformational change in Glu-plasminogen distinct from that of Lys-Pg.

uPA deficiency exacerbates muscular dystrophy in MDX mice.

Duchenne muscular dystrophy (DMD) is a fatal and incurable muscle degenerative disorder. We identify a function of the protease urokinase plasminogen activator (uPA) in mdx mice, a mouse model of DMD. The expression of uPA is induced in mdx dystrophic muscle, and the genetic loss of uPA in mdx mice exacerbated muscle dystrophy and reduced muscular function. Bone marrow (BM) transplantation experiments revealed a critical function for BM-derived uPA in mdx muscle repair via three mechanisms: (1) by promoting the infiltration of BM-derived inflammatory cells; (2) by preventing the excessive deposition of fibrin; and (3) by promoting myoblast migration. Interestingly, genetic loss of the uPA receptor in mdx mice did not exacerbate muscular dystrophy in mdx mice, suggesting that uPA exerts its effects independently of its receptor. These findings underscore the importance of uPA in muscular dystrophy.

Characterization of plasminogen binding to NB4 promyelocytic cells using monoclonal antibodies against receptor-induced binding sites in cell-bound plasminogen.

The NB4 promyelocytic cell line exhibits many of the characteristics of acute promyelocytic leukemia blast cells, including the translocation (15 : 17) that fuses the PML gene on chromosome 15 to the RARα gene on chromosome 17. These cells have a very high fibrinolytic capacity. In addition to a high secretion of urokinase, NB4 cells exhibit a 10-fold higher plasminogen binding capacity compared with other leukemic cell lines. When tissue-type plasminogen activator was added to acid-treated cells, plasmin generation was 20-26-fold higher than that generated by U937 cells or peripheral blood neutrophils, respectively. We found that plasminogen bound to these cells can be detected by fluorescence-activated cell sorting using an antiplasminogen monoclonal antibody that specifically reacts with this antigen when it is bound to cell surfaces. All-trans retinoid acid treatment of NB4 cells markedly decreased the binding of this monoclonal antibody. This cell line constitutes a unique model to explore plasminogen binding and activation on cell surfaces that can be modulated by all-trans retinoid acid treatment.

Sestrin prevents atrophy of disused and aging muscles by integrating anabolic and catabolic signals.

A unique property of skeletal muscle is its ability to adapt its mass to changes in activity. Inactivity, as in disuse or aging, causes atrophy, the loss of muscle mass and strength, leading to physical incapacity and poor quality of life. Here, through a combination of transcriptomics and transgenesis, we identify sestrins, a family of stress-inducible metabolic regulators, as protective factors against muscle wasting. Sestrin expression decreases during inactivity and its genetic deficiency exacerbates muscle wasting; conversely, sestrin overexpression suffices to prevent atrophy. This protection occurs through mTORC1 inhibition, which upregulates autophagy, and AKT activation, which in turn inhibits FoxO-regulated ubiquitin-proteasome-mediated proteolysis. This study reveals sestrin as a central integrator of anabolic and degradative pathways preventing muscle wasting. Since sestrin also protected muscles against aging-induced atrophy, our findings have implications for sarcopenia.

FoxO maintains a genuine muscle stem-cell quiescent state until geriatric age.

Tissue regeneration declines with ageing but little is known about whether this arises from changes in stem-cell heterogeneity. Here, in homeostatic skeletal muscle, we identify two quiescent stem-cell states distinguished by relative CD34 expression: CD34High, with stemness properties (genuine state), and CD34Low, committed to myogenic differentiation (primed state). The genuine-quiescent state is unexpectedly preserved into later life, succumbing only in extreme old age due to the acquisition of primed-state traits. Niche-derived IGF1-dependent Akt activation debilitates the genuine stem-cell state by imposing primed-state features via FoxO inhibition. Interventions to neutralize Akt and promote FoxO activity drive a primed-to-genuine state conversion, whereas FoxO inactivation deteriorates the genuine state at a young age, causing regenerative failure of muscle, as occurs in geriatric mice. These findings reveal transcriptional determinants of stem-cell heterogeneity that resist ageing more than previously anticipated and are only lost in extreme old age, with implications for the repair of geriatric muscle.

HIV-1 transgenic expression in mice induces selective atrophy of fast-glycolytic skeletal muscle fibers.

Human immunodeficiency virus (HIV)-induced wasting syndrome, characterized by weakness and severe loss of muscle mass, is a common condition of patients with advanced acquired immunodeficiency syndrome (AIDS). The homozygous HIV-1 transgenic mouse line Tg26 reproduces the wasting syndrome of AIDS patients, thus constituting a valid animal model to characterize the muscle phenotype induced by HIV infection. In this study, we identified a selective atrophy of fast-glycolytic myofibers in skeletal muscles of homozygous HIV-1 transgenic mice, whereas the more oxidative fiber types are spared. In agreement with this, muscles enriched in fast-glycolytic myofibers such as the extensor digitorum longus and gastrocnemius, but not those rich in oxidative fibers such as the soleus, exhibited a reduced muscle size in homozygous HIV-1 transgenic mice compared to their littermate control counterparts. Additionally, muscles of heterozygous HIV-1 transgenic mice displayed increased inflammation and blunted myofiber growth in an injury-induced muscle regeneration process. Since no myogenic intrinsic defect was observed in satellite cells from the transgenic mice, these results support the notion of an inflammation-mediated, fiber-type-specific inhibition of muscle growth in the presence of the HIV-1 transgene.

The plasminogen activation system in skeletal muscle regeneration: antagonistic roles of urokinase-type plasminogen activator (uPA) and its inhibitor (PAI-1).

The plasminogen activation (PA) system is an extensively used mechanism for the generation of proteolytic activity in the extracellular matrix, where it contributes to tissue remodeling in a wide range of physiopathological processes. Despite the limited information available at present on plasminogen activators, their inhibitors and cognate receptors in skeletal muscle, increasing evidence is accumulating on their important roles in the homeostasis of muscle fibers and their surrounding extracellular matrix. The development of mice deficient for the individual components of the PA system has provided an incisive approach to test the proposed muscle functions in vivo. Skeletal muscle regeneration induced by injury has been analyzed in urokinase-type plasminogen activator (uPA)-, tissue-type plasminogen activator (tPA)-, plasminogen (Plg)- and plasminogen activator inhibitor-1 (PAI-1)-deficient mice and has demonstrated profound effects of these molecules on the fibrotic state and the inflammatory response, which contribute to muscle repair. In particular, the opposite roles of uPA and its inhibitor PAI-1 in this process are highlighted. Delineating the mechanisms by which the different plasminogen activation system components regulate tissue repair will be of potential therapeutic value for severe muscle disorders.

The alkylating carcinogen N-methyl-N'-nitro-N-nitrosoguanidine activates the plasminogen activator inhibitor-1 gene through sequential phosphorylation of p53 by ATM and ATR kinases.

The alkylating agent MNNG is an environmental carcinogen that causes DNA lesions leading to cell death. We previously demonstrated that MNNG induced the transcriptional activity of the plasminogen activator inhibitor-1 (PAI-1) gene in a p53-dependent manner. However, the mechanism(s) linking external MNNG stimulation and PAI-1 gene induction remained to be elucidated. Here, we show that ATM and ATR kinases, but not DNA-PK, which participate in DNA damage-activated checkpoints, regulate the phosphorylation of p53 at serine 15 in response to MNNG cell treatment. Using ATM-deficient cells, ATM was shown to be required for early phosphorylation of serine 15 in response to MNNG, whereas catalytically inactive ATR selectively interfered with late phase serine 15 phosphorylation. In contrast, DNA-PK-deficient cells showed no change in the MNNG-induced serine 15 phosphorylation pattern. In agreement with this, sequential activation of ATM and ATR kinases was also required for adequate induction of the endogenous PAI-1 gene by MNNG. Finally, we showed that cells derived from PAI-1-deficient mice were more resistant to MNNG-induced cell death than normal cells, suggesting that p53-dependent PAI-1 expression partially mediated this effect. Since PAI-1 is involved in the control of tumor invasiveness, our finding that MNNG induces PAI-1 gene expression via ATM/ATR-mediated phosphorylation of p53 sheds new insight on the role of these DNA damage-induced cell cycle checkpoint kinases.

Fibrogenic Cell Plasticity Blunts Tissue Regeneration and Aggravates Muscular Dystrophy.

Preservation of cell identity is necessary for homeostasis of most adult tissues. This process is challenged every time a tissue undergoes regeneration after stress or injury. In the lethal Duchenne muscular dystrophy (DMD), skeletal muscle regenerative capacity declines gradually as fibrosis increases. Using genetically engineered tracing mice, we demonstrate that, in dystrophic muscle, specialized cells of muscular, endothelial, and hematopoietic origins gain plasticity toward a fibrogenic fate via a TGFß-mediated pathway. This results in loss of cellular identity and normal function, with deleterious consequences for regeneration. Furthermore, this fibrogenic process involves acquisition of a mesenchymal progenitor multipotent status, illustrating a link between fibrogenesis and gain of progenitor cell functions. As this plasticity also was observed in DMD patients, we propose that mesenchymal transitions impair regeneration and worsen diseases with a fibrotic component.

p38/MKP-1-regulated AKT coordinates macrophage transitions and resolution of inflammation during tissue repair.

Repair of damaged tissue requires the coordinated action of inflammatory and tissue-specific cells to restore homeostasis, but the underlying regulatory mechanisms are poorly understood. In this paper, we report new roles for MKP-1 (mitogen-activated protein kinase [MAPK] phosphatase-1) in controlling macrophage phenotypic transitions necessary for appropriate muscle stem cell-dependent tissue repair. By restricting p38 MAPK activation, MKP-1 allows the early pro- to antiinflammatory macrophage transition and the later progression into a macrophage exhaustion-like state characterized by cytokine silencing, thereby permitting resolution of inflammation as tissue fully recovers. p38 hyperactivation in macrophages lacking MKP-1 induced the expression of microRNA-21 (miR-21), which in turn reduced PTEN (phosphatase and tensin homologue) levels, thereby extending AKT activation. In the absence of MKP-1, p38-induced AKT activity anticipated the acquisition of the antiinflammatory gene program and final cytokine silencing in macrophages, resulting in impaired tissue healing. Such defects were reversed by temporally controlled p38 inhibition. Conversely, miR-21-AKT interference altered homeostasis during tissue repair. This novel regulatory mechanism involving the appropriate balance of p38, MKP-1, miR-21, and AKT activities may have implications in chronic inflammatory degenerative diseases.

Efficient adult skeletal muscle regeneration in mice deficient in p38beta, p38gamma and p38delta MAP kinases.

Adult skeletal muscle is a very stable tissue containing a small population of myofiber-associated quiescent satellite cells compared with late embryonic/neonatal skeletal muscle, which contains highly proliferating myoblasts and small actively growing myofibers, suggesting that specific regulatory pathways may control myogenesis at distinct developmental stages. The p38 MAPK signaling pathway is central for myogenesis, based on studies using immortalized and neonatal primary myoblasts in vitro. However, the contribution of this pathway to adult myogenesis has never been investigated. Four p38 isoforms (p38alpha, p38beta, p38gamma and p38delta) exist in mammalian cells, being p38alpha and p38gamma the most abundantly expressed isoforms in adult skeletal muscle. Given the embryonic/neonatal lethality of p38alpha-deficient mice, here we investigate the relative contribution of p38beta, p38gamma and p38delta to adult myogenesis. Regeneration and myofiber growth of adult muscle proceeds with similar efficiency in mice lacking p38beta, p38gamma and p38delta as in wild-type control mice. In agreement with this, there is no difference in adult primary myoblasts behavior in vitro among the different genotypes. Importantly, the pattern of p38 activation (ascribed to p38alpha) remains unperturbed during satellite cell-mediated myogenesis in vitro and adult muscle regeneration in wild type and p38beta-, p38gamma- and p38delta-deficient mice, rendering p38alpha as the essential p38 isoform sustaining adult myogenesis. This study constitutes the first analysis addressing the functionality of p38beta, p38gamma and p38delta in satellite cell-dependent adult muscle regeneration and growth.

Fibrinogen drives dystrophic muscle fibrosis via a TGFbeta/alternative macrophage activation pathway.

In the fatal degenerative Duchenne muscular dystrophy (DMD), skeletal muscle is progressively replaced by fibrotic tissue. Here, we show that fibrinogen accumulates in dystrophic muscles of DMD patients and mdx mice. Genetic loss or pharmacological depletion of fibrinogen in these mice reduced fibrosis and dystrophy progression. Our results demonstrate that fibrinogen-Mac-1 receptor binding, through induction of IL-1beta, drives the synthesis of transforming growth factor-beta (TGFbeta) by mdx macrophages, which in turn induces collagen production in mdx fibroblasts. Fibrinogen-produced TGFbeta further amplifies collagen accumulation through activation of profibrotic alternatively activated macrophages. Fibrinogen, by engaging its alphavbeta3 receptor on fibroblasts, also directly promotes collagen synthesis. These data unveil a profibrotic role of fibrinogen deposition in muscle dystrophy.

Genetic analysis of p38 MAP kinases in myogenesis: fundamental role of p38alpha in abrogating myoblast proliferation.

The p38 mitogen-activated protein kinase (MAPK) pathway plays a critical role in skeletal muscle differentiation. However, the relative contribution of the four p38 MAPKs (p38alpha, p38beta, p38gamma and p38delta) to this process is unknown. Here we show that myoblasts lacking p38alpha, but not those lacking p38beta or p38delta, are unable to differentiate and form multinucleated myotubes, whereas p38gamma-deficient myoblasts exhibit an attenuated fusion capacity. The defective myogenesis in the absence of p38alpha is caused by delayed cell-cycle exit and continuous proliferation in differentiation-promoting conditions. Indeed, activation of JNK/cJun was enhanced in p38alpha-deficient myoblasts leading to increased cyclin D1 transcription, whereas inhibition of JNK activity rescued the proliferation phenotype. Thus, p38alpha controls myogenesis by antagonizing the activation of the JNK proliferation-promoting pathway, before its direct effect on muscle differentiation-specific gene transcription. More importantly, in agreement with the defective myogenesis of cultured p38alpha(Delta/Delta) myoblasts, neonatal muscle deficient in p38alpha shows cellular hyperproliferation and delayed maturation. This study provides novel evidence of a fundamental role of p38alpha in muscle formation in vitro and in vivo.

Inhibition of cell surface mediated plasminogen activation by a monoclonal antibody against alpha-Enolase.

Localization of plasmin activity on leukocyte surfaces plays a critical role in fibrinolysis as well as in pathological and physiological processes in which cells must degrade the extracellular matrix in order to migrate. The binding of plasminogen to leukocytic cell lines induces a 30- to 80-fold increase in the rate of plasminogen activation by tissue-type (tPA) and urokinase-type (uPA) plasminogen activators. In the present study we have examined the role of alpha-enolase in plasminogen activation on the cell surface. We produced and characterized a monoclonal antibody (MAb) 11G1 against purified alpha-enolase, which abrogated about 90% of cell-dependent plasminogen activation by either uPA or tPA on leukocytoid cell lines of different lineages: B-lymphocytic, T-lymphocytic, granulocytic, and monocytic cells. In addition, MAb 11G1 also blocked enhancement of plasmin formation by peripheral blood neutrophils and monocytes. In contrast, MAb 11G1 did not affect plasmin generation in the presence of fibrin, indicating that this antibody did not interact with fibrinolytic components in the absence of cells. These data suggest that, although leukocytic cells display several molecules that bind plasminogen, alpha-enolase is responsible for the majority of the promotion of plasminogen activation on the surfaces of leukocytic cells.