Satellite Cell Apoptosis Research Articles

Increased satellite cell apoptosis in vastus lateralis muscle after anterior cruciate ligament reconstruction.

ObjectiveRecovery of the quadriceps femoris muscle after anterior ligament reconstruction is impaired. The aim of this study was to investigate satellite cell content and function of the vastus lateralis muscle after anterior ligament reconstruction.MethodsBiopsies were obtained from the vastus lateralis muscle of 16 recreational athletes immediately before and again 12 weeks after anterior ligament reconstruction. Total satellite cell number (Pax7+), activated (Pax7+/MyoD+), differentiating (Pax7–/MyoD+), and apoptotic (Pax7+/TUNEL+) satellite cells, myofibers expressing myosin heavy chain (MHC) I and II, and neonatal MHC (MHCneo) were determined immunohistochemically.ResultsAfter anterior ligament reconstruction, the number of apoptotic satellite cells was significantly (p = 0.019) increased, concomitant with a significant (p < 0.001) decrease in total satellite cell number, with no change in activated and differentiating satellite cell number. MHCneo+ myofibers tended towards an increase.CONCLUSIONSatellite cell apoptosis and the reduction in the satellite cell pool might provide an explanation for prolonged quadriceps muscle atrophy after anterior ligament reconstruction.LAY ABSTRACTProtracted muscle atrophy is common after anterior ligament reconstruction, even if athletes adhere to a structured rehabilitation programme. Satellite cells, the stem cells of skeletal muscle, play an important role in recovery of an atrophied muscle. Exercise can activate satellite cells, induce their proliferation, and probably also differentiation of these stem cells. The current study evaluated satellite cell content and function in biopsies from the vastus lateralis muscle of 16 recreational athletes immediately before and 12 weeks after anterior ligament reconstruction. After anterior ligament reconstruction, an increased number of satellite cells showed signs of apoptosis (cell death). Furthermore, total satellite cell number was decreased, with no change in the numbers of activated and differentiating satellite cells. The number of regenerating myofibers expressing neonatal myosin tended to increase. In conclusion, satellite cell apoptosis and the reduced satellite cell number might provide an explanation for the impaired muscle recovery after anterior ligament reconstruction.

PSIX-29 JAK2-STAT3 Pathway Mediated Satellite Cell Apoptosis to Govern Skeletal Muscle Growth with Lysine

Abstract Apoptosis is programmed cell death that can be stimulated by external stress or nutrition restrictions. Lysine (Lys) is an essential amino acid for pig growth, and the relationship between Lys deficiency caused apoptosis and inhibition of skeletal muscle growth remains unknown. The objective of this study was to investigate whether apoptosis could be regulated by Lys supplementation and the potential mechanism. In current work, 30 male Duroc × Landrace × Large weaned piglets were divided randomly into 3 groups: control group (Lys 1.30%), Lys deficiency group (Lys 0.86%), and Lys rescue group (Lys 0.86%, 0-14d; 1.30%,15–28 d). The experiment lasted for 28 days, and on the morning of 29 d, piglets were slaughtered to collect samples. Isobaric tag for relative and absolute quantification (iTRAQ) proteomics analysis of the longissimus dorsi muscle showed that Janus family tyrosine kinase (JAK)-signal transducer and activator of transcription (STAT) pathway was involved in Lys deficiency-induced apoptosis and inhibited skeletal muscle growth. Meanwhile, western blotting results of the longissimus dorsi muscle demonstrated that Lys deficiency caused apoptosis (P &amp;lt; 0.05) with the JAK2-STAT3 pathway inhibition (P &amp;lt; 0.05). Interestingly, apoptosis was suppressed (P &amp;lt; 0.05), and the JAK2-STAT3 pathway was reactivated (P &amp;lt; 0.05) after Lys re-supplementation in longissimus dorsi muscle. In addition, results of satellite cells (SCs) isolated from the longissimus dorsi muscle of 5-day-old Landrace piglets showed that Lys deficiency-induced apoptosis (P &amp;lt; 0.05) was mediated by the JAK2-STAT3 pathway inhibition (P &amp;lt; 0.05). Moreover, the JAK2-STAT3 pathway was reactivated (P &amp;lt; 0.05) by Lys re-supplementation and suppressed apoptosis in SCs (P &amp;lt; 0.05), and this effect was blocked (P &amp;lt; 0.05) after SCs treated with AG-490 (a specific inhibitor of JAK2). Collectively, Lys inhibited apoptosis in SCs to govern skeletal muscle growth via the JAK2-STAT3 pathway.

Estrogen Receptor β Controls Muscle Growth and Regeneration in Young Female Mice.

SummaryEstrogens are female sex hormones that are important for comprehensively maintaining muscle function, and an insufficiency affects muscle strength and regeneration in females. However, it is still unclear whether estrogen signaling is mediated through receptors. To investigate the specific role of estrogen receptor β (ERβ) in skeletal muscle and satellite cells (muscle stem cells), we generated muscle-specific ERβ-knockout (mKO) and satellite cell-specific ERβ-knockout (scKO) mice, respectively. Young female mKO mice displayed a decrease in fast-type dominant muscle mass. Female, but not male, scKO mice exhibited impaired muscle regeneration following acute muscle injury, probably due to reduced proliferation and increased apoptosis of satellite cells. RNA-sequencing analysis revealed that loss of ERβ in satellite cells altered gene expression of extracellular matrix components, including laminin and collagen. The results indicate that the estrogen-ERβ pathway is a sex-specific regulatory mechanism that controls muscle growth and regeneration in female mice.

Estrogen Regulates the Satellite Cell Compartment in Females

SUMMARYSkeletal muscle mass, strength, and regenerative capacity decline with age, with many measures showing a greater deterioration in females around the time estrogen levels decrease at menopause. Here, we show that estrogen deficiency severely compromises the maintenance of muscle stem cells (i.e., satellite cells) as well as impairs self-renewal and differentiation into muscle fibers. Mechanistically, by hormone replacement, use of a selective estrogen-receptor modulator (bazedoxifene), and conditional estrogen receptor knockout, we implicate 17β-estradiol and satellite cell expression of estrogen receptor α and show that estrogen signaling through this receptor is necessary to prevent apoptosis of satellite cells. Early data from a biopsy study of women who transitioned from peri- to post-menopause are consistent with the loss of satellite cells coincident with the decline in estradiol in humans. Together, these results demonstrate an important role for estrogen in satellite cell maintenance and muscle regeneration in females.

Forkhead Box M1 Transcriptionally Regulates the Expression of Long Noncoding RNAs Snhg8 and Gm26917 to Promote Proliferation and Survival of Muscle Satellite Cells.

Multiple functions have been proposed for transcription factor FoxM1, including the regulation of cell proliferation, differentiation, senescence, apoptosis, and tissue homeostasis. However, the role of FoxM1 in muscle satellite cells (SCs) remains unclear. In the present study, we demonstrated that FoxM1 was essential for the proliferation and survival of SCs. Crucially, we found that long noncoding RNAs (lncRNAs) Snhg8 and Gm26917 significantly regulated the proliferation and apoptosis of SCs, respectively, and these lncRNAs were directly regulated by FoxM1 in SCs. Mechanistically, Snhg8 sustained SCs proliferation by promoting the transcription of ribosomal proteins, while Gm26917 acted as a competing endogenous RNA for microRNA-29b, which accelerated apoptosis of SCs. In mice, conditional knockout of FoxM1 in skeletal muscle resulted in decreased proliferation and increased apoptosis of SCs. Thus, our studies revealed a previously unrecognized role of FoxM1 in SCs and uncovered two lncRNAs, Snhg8 and Gm26917, which function as novel targets of FoxM1 in the regulation of SCs proliferation and survival. Stem Cells 2018;36:1097-1108.

Estrogen Regulates the Satellite Cell Compartment in Females

Skeletal muscle mass, strength, and regenerative capacity decline with age, with many measures showing greater deterioration in females about the time estrogen levels decrease at menopause. Here we show that maintenance of muscle stem cells, satellite cells, as well as self-renewal and differentiation into muscle fibers, are severely compromised by estrogen deficiency. Mechanistically, by hormone replacement, use of a selective estrogen-receptor modulator (bazedoxifene), and conditional estrogen receptor knockout, we implicate 17β-estradiol and satellite cell expression of estrogen receptor alpha (ERalpha) and show that estrogen signaling through this receptor is necessary to prevent apoptosis of satellite cells. Early data from a biopsy study of women who transitioned from peri- to post-menopause are consistent with the loss of satellite cells coincident with the decline in estradiol in humans. Together, these results demonstrate an important role for estrogen in satellite cell maintenance and muscle regeneration in females.

Satellite cell activation and apoptosis in skeletal muscle from severely burned children.

Severe burns result in profound skeletal muscle atrophy that hampers recovery. The activity of skeletal muscle stem cells, satellite cells, acutely following a severe burn is unknown and may contribute to the recovery of lean muscle. Severe burn injury induces skeletal muscle regeneration and myonuclear apoptosis. Satellite cells undergo concurrent apoptosis and activation acutely following a burn, with a net reduction in satellite cell content compared to healthy controls. The activation and apoptosis of satellite cells probably impacts the recovery of lean tissue following a severe burn, contributing to prolonged frailty in burn survivors. Severe burns result in profound skeletal muscle atrophy; persistent muscle loss and weakness are major complications that hamper recovery from burn injury. Many factors contribute to the erosion of muscle mass following burn trauma and we propose that an impaired muscle satellite cell response is key in the aetiology of burn-induced cachexia. Muscle biopsies from the m. vastus lateralis were obtained from 12 male pediatric burn patients (>30% total body surface area burn) and 12 young, healthy male subjects. Satellite cell content, activation and apoptosis were determined via immunohistochemistry, as were muscle fibre regeneration and myonuclear apoptosis. Embryonic myosin heavy chain expression and central nucleation, indices of skeletal muscle regeneration, were elevated in burn patients (P<0.05). Myonuclear apoptosis, quantified by TUNEL positive myonuclei and cleaved caspase-3 positive myonuclei, was also elevated in burn patients (P<0.05). Satellite cell content was reduced in burn patients, with approximately 20% of satellite cells positive for TUNEL staining, indicating DNA damage associated with apoptosis (P<0.05). Additionally, a significant percentage of satellite cells in burn patients expressed Ki67, a marker for cellular proliferation (P<0.05). Satellite cell activation was also observed in burn patients with increased expression of MyoD compared to healthy controls (P<0.05). Robust skeletal muscle atrophy occurs after burn injury, even in muscles located distally to the site of injury. The activation and apoptosis of satellite cells probably impacts the recovery of lean tissue following a severe burn, contributing to prolonged frailty in burn survivors.

Intracellular Inactivation of Thyroid Hormone Is a Survival Mechanism for Muscle Stem Cell Proliferation and Lineage Progression

SummaryPrecise control of the thyroid hormone (T3)-dependent transcriptional program is required by multiple cell systems, including muscle stem cells. Deciphering how this is achieved and how the T3 signal is controlled in stem cell niches is essentially unknown. We report that in response to proliferative stimuli such as acute skeletal muscle injury, type 3 deiodinase (D3), the thyroid hormone-inactivating enzyme, is induced in satellite cells where it reduces intracellular thyroid signaling. Satellite cell-specific genetic ablation of dio3 severely impairs skeletal muscle regeneration. This impairment is due to massive satellite cell apoptosis caused by exposure of activated satellite cells to the circulating TH. The execution of this proapoptotic program requires an intact FoxO3/MyoD axis, both genes positively regulated by intracellular TH. Thus, D3 is dynamically exploited in vivo to chronically attenuate TH signaling under basal conditions while also being available to acutely increase gene programs required for satellite cell lineage progression.

The effect of nutritional status and muscle fiber type on myogenic satellite cell fate and apoptosis

Satellite cells (SC) are multipotential stem cells that can be induced by nutrition to alter their cellular developmental fate, which may vary depending on their fiber type origin. The objective of the current study was to determine the effect of restricting protein synthesis on inducing adipogenic transdifferentiation and apoptosis of SC originating from fibers of the fast glycolytic pectoralis major (p. major) and fast oxidative and glycolytic biceps femoris (b. femoris) muscles of the chicken. The availability of the essential sulfur amino acids Met and Cys was restricted to regulate protein synthesis during SC proliferation and differentiation. The SC were cultured and treated with 1 of 6 Met/Cys concentrations: 60/192, 30/96 (control), 7.5/24, 3/9.6, 1/3.2, or 0/0 mg/L. Reductions in Met/Cys concentrations from the control level resulted in increased lipid staining and expression of the adipogenic marker genes peroxisome proliferator-activated receptor gamma and stearoyl-CoA desaturase during differentiation in the p. major SC. Although b. femoris SC had increased lipid staining at lower Met/Cys concentrations, there was no increase in expression of either adipogenic gene. For both muscle types, SC Met/Cys, concentration above the control increased the expression of peroxisome proliferator-activated receptor gamma and stearoyl-CoA desaturase during differentiation. As Met/Cys concentration was decreased during proliferation, a dose-dependent decline in all apoptotic cells occurred except for early apoptotic cells in the p. major, which had no treatment effect (P < 0.05). During differentiation, decreasing Met/Cys concentration caused an increase in early apoptotic cells in both fiber types and no effect on late apoptotic cells except for an increase in the p. major 7.5/24 mg/L of Met/Cys treatment. In general, the viability of the SC was unaffected by the Met/Cys concentration except during proliferation in the p. major 0/0 mg/L of Met/Cys treatment, which increased SC viability. These data demonstrate the effect of nutrition on SC transdifferentiation to an adipogenic lineage and apoptosis, and the effect of fiber type on this response in an in vitro context.

Involvement of the caspase enzymes in satellite cells apoptosis

A is characterized by an impairment of muscle regenerative potential and a progressive loss of skeletal muscle. This condition, called sarcopenia, has important health care implications for humans, as it contributes to frailty, functional loss and premature death. The ability of skeletal muscle to regenerate is owed to a population of myogenic stem cells called satellite cells (SCs). In previous papers of our research group we demonstrated an age-related decrease in the antioxidant capacity of human SCs that may negatively affect the ageing SCs ability to repair muscle. Despite the involvement of caspases in muscle wasting, the real role of these enzymes is still controversial. To test the possibility of caspases involvement in SCs death in human ageing muscles, we cultured in vitro primary cells derived from vastus lateralis of young and aged subjects. We analysed both initiator caspases and effector caspases in the presence or absence of specific or broad pharmacologic inhibitors. Furthermore, we evaluated the expression of various genes that play a critical role in oxidative stress and cell death. Our findings highlighted an increased rate of spontaneous apoptosis and an up-regulation of CASP2, 6, 7, 9 and other cell death genes in aged SCs, supporting the hypothesis of an intrinsic ageing of SCs and previous reports demonstrating an increased susceptibility to apoptosis of SCs from old animals. These results suggest that a greater proportion of SCs from old subjects might undergo programmed cell death in vivo in response to stressful stimuli, thereby impairing skeletal muscle regeneration.

Apoptosis in capillary endothelial cells in ageing skeletal muscle

The age-related loss of skeletal muscle mass and function (sarcopenia) is a consistent hallmark of ageing. Apoptosis plays an important role in muscle atrophy, and the intent of this study was to specify whether apoptosis is restricted to myofibre nuclei (myonuclei) or occurs in satellite cells or stromal cells of extracellular matrix (ECM). Sarcopenia in mouse gastrocnemius muscle was characterized by myofibre atrophy, oxidative type grouping, delocalization of myonuclei and ECM fibrosis. Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) indicated a sharp rise in apoptosis during ageing. TUNEL coupled with immunostaining for dystrophin, paired box protein-7 (Pax7) or laminin-2α, respectively, was used to identify apoptosis in myonuclei, satellite cells and stromal cells. In adult muscle, apoptosis was not detected in myofibres, but was restricted to stromal cells. Moreover, the age-related rise in apoptotic nuclei was essentially due to stromal cells. Myofibre-associated apoptosis nevertheless occurred in old muscle, but represented < 20% of the total muscle apoptosis. Specifically, apoptosis in old muscle affected a small proportion (0.8%) of the myonuclei, but a large part (46%) of the Pax7+ satellite cells. TUNEL coupled with CD31 immunostaining further attributed stromal apoptosis to capillary endothelial cells. Age-dependent rise in apoptotic capillary endothelial cells was concomitant with altered levels of key angiogenic regulators, perlecan and a perlecan domain V (endorepellin) proteolytic product. Collectively, our results indicate that sarcopenia is associated with apoptosis of satellite cells and impairment of capillary functions, which is likely to contribute to the decline in muscle mass and functionality during ageing.

Function of death-associated protein 1 in proliferation, differentiation, and apoptosis of chicken satellite cells

Muscle growth and regeneration are processes closely associated with proliferation, differentiation, and apoptosis of muscle cells. Death-associated protein 1 (DAP1) has been identified as a negative regulator of autophagy. Little is known about the function of DAP1 in the regulation of myogenesis and satellite cells. Chicken satellite cells were transfected with DAP1 cloned into the pCMS-enhanced green fluorescent protein vector or pcDNA3.1 vector, or a small interference RNA against the endogenous DAP1 gene. The cells were assayed for proliferation, differentiation, and apoptosis. The overexpression of DAP1 increased proliferation, differentiation, and myotube diameter, but it had no effect on satellite cell apoptosis. In contrast, knockdown of DAP1 significantly decreased proliferation, differentiation, and number of nuclei per myotube, and it increased apoptosis of the cells. DAP1 is required for regulating myogenesis and apoptosis of satellite cells, which may affect muscle mass accretion and regeneration, and ameliorate muscle sarcopenia.

Role of syndecan-4 side chains in turkey satellite cell apoptosis and focal adhesion formation

S4 (syndecan-4) is a cell membrane heparan sulfate proteoglycan that functions in muscle growth and development. It is composed of a central core protein and two types of side chains: GAGs (glycosaminoglycans) and N-glycosylated (N-linked glycosylated) chains. The N-glycosylated chains and GAG chains are required for S4 to regulate turkey myogenic satellite cell proliferation. The objective of the current study was to determine whether the S4 side chains regulate cell proliferation through muscle cell focal adhesion formation and apoptosis. S4 mutants with only one or without any N-glycosylated chains attached to the core protein with or without GAG chains were generated to study the function of N-glycosylated chains and the interaction between N-glycosylated chains and GAG chains. The wild-type S4 and all of the S4 side chain mutants were transfected into turkey myogenic satellite cells. Cell apoptosis and focal adhesion formation were measured, and PKCα (protein kinase Cα) cell membrane localization was investigated. S4 increased FAK (focal adhesion kinase) activity and the deletion of the side chains decreased this effect. S4 and the S4 mutants had no effect on β1-integrin expression, but increased the cell membrane localization of β1-integrin and PKCα. Furthermore, cell apoptosis and vinculin containing focal adhesions were not affected by S4 and its mutants. The results suggest that S4 and its side chains play important roles in regulating FAK activity, and PKCα and β1-integrin cell membrane localization, but not cell apoptosis and vinculin-containing focal adhesion formation.

Defective Neuromuscular Junction Organization and Postnatal Myogenesis in Mice With Severe Spinal Muscular Atrophy

A detailed pathologic analysis was performed on Smn(-/-);SMN2 mice as a mouse model for human type I spinal muscular atrophy (SMA). We provide new data concerning changes in the spinal cord, neuromuscular junctions and muscle cells, and in the organs of the immune system. The expression of 10 synaptic proteins was analyzed in 3-dimensionally reconstructed neuromuscular junctions by confocal microscopy. In addition to defects in postsynaptic occupancy, there was a marked reduction in calcitonin gene-related peptide and Rab3A in the presynaptic motor terminals of some, but not all, of the skeletal muscles analyzed. Defects in the organization of presynaptic nerve terminals were also detected by electron microscopy. Moreover, degenerative changes in muscle cells, defective postnatal muscle growth, and prominent muscle satellite cell apoptosis were also observed. All of these changes occurred in the absence of massive loss of spinal cord motoneurons. On the other hand, astroglia, but not microglia, increased in the ventral horn of newborn SMA mice. In skeletal muscles, the density of interstitial macrophages was significantly reduced, and monocyte chemotactic protein-1 was downregulated. These findings raise questions regarding the primary contribution of a muscle cell defect to the SMA phenotype.

The role of systemic inflammation in age‐related muscle weakness and wasting

Ageing is associated with a slow, but progressive muscle weakness, which is largely attributable to muscle wasting. A diminished function of satellite cells at old age may hamper preservation and repair from (contraction)-induced injury and contribute to the age-related muscle wasting. Satellite cell function may be affected by circulating factors, as muscle regeneration in old mice sharing the circulation of young mice is not impaired. Chronic low-grade systemic inflammation in old organisms may be that environmental factor. Indeed, the inflammatory cytokine tumor necrosis factor-alpha (TNFalpha) negatively affects the muscle regenerating capacity. TNFalpha destabilizes MyoD, a muscle-specific transcription factor involved in satellite cell proliferation and differentiation, and induces apoptosis of satellite cells, particularly at old age. Here it is proposed that some of these effects are mediated by TNFalpha-induced expression of inhibitors of differentiation proteins. Yet, the increase in TNFalpha during the normal inflammatory response helps, rather than impairs, the repair process. This apparent contradiction may be resolved by the fact that the effects of TNFalpha are concentration and time dependent. Thus, the negative effect of systemic inflammation on muscle strength at old age may only become apparent when it exceeds a certain threshold and persists for a prolonged period.

Transforming growth factor-β1-induced satellite cell apoptosis in chickens is associated with β1 integrin-mediated focal adhesion kinase activation

Transforming growth factor-β1 (TGF-β1) induces apoptosis in many types of cells. The cell adhesion receptor, β1 integrin subunit, prevents apoptosis and may be involved in TGF-β1-induced muscle cell apoptosis. In the current study, chicken primary satellite cells, myogenic precursors, were used to investigate the apoptotic effect of TGF-β1 on muscle cells. The data from the current study showed that the addition of exogenous TGF-β1 reduced β1 integrin expression and altered its localization. Treatment of the satellite cells with TGF-β1 increased the number of apoptotic cells indicated by annexin-V using flow cytometry. The number of caspase-positive cells was increased in the TGF-β1-treated immunostained cells, which supported that TGF-β1 induced satellite cell apoptosis. It has been shown that β1 integrin is involved in muscle cell survival. In response to the activation of β1 integrin, focal adhesion kinase (FAK) phosphorylates tyrosine at residue 397 and activates cell survival signal transduction. The phosphorylation of FAK was significantly reduced from 30 min to 4 h after TGF-β1 treatment during both satellite cell proliferation and differentiation. These data suggested that the apoptotic effect of TGF-β1 on satellite cells is likely associated with a β1 integrin-mediated FAK signaling pathway during satellite cell proliferation and differentiation.

Skeletal muscle stem cells express anti-apoptotic ErbB receptors during activation from quiescence

To be effective for tissue repair, satellite cells (the stem cells of adult muscle) must survive the initial activation from quiescence. Using an in vitro model of satellite cell activation, we show that erbB1, erbB2 and erbB3, members of the EGF receptor tyrosine kinase family, appear on satellite cells within 6 h of activation. We show that signalling via erbB2 provides an anti-apoptotic survival mechanism for satellite cells during the first 24 h, as they progress to a proliferative state. Inhibition of erbB2 signalling with AG825 reduced satellite cell numbers, concomitant with elevated caspase-8 activation and TUNEL labelling of apoptotic satellite cells. In serum-free conditions, satellite cell apoptosis could be largely prevented by a mixture of erbB1, erbB3 and erbB4 ligand growth factors, but not by neuregulin alone (erbB3/erbB4 ligand). Furthermore, using inhibitors specific to discrete intracellular signalling pathways, we identify MEK as a pro-apoptotic mediator, and the erbB-regulated factor STAT3 as an anti-apoptotic mediator during satellite cell activation. These results implicate erbB2 signalling in the preservation of a full compliment of satellite cells as they activate in the context of a damaged muscle.

Proapoptotic factor Bax is increased in satellite cells in the tibialis anterior muscles of old rats

Aging impairs the ability of muscle to adapt to exercise or injury. The goal of this study was to determine whether age-related changes in muscle adaptability could be the result of satellite cell apoptosis. Ten days after exposure to an injury protocol, estimates of edema in the exposed tibialis anterior muscles were higher in old (30 months) than young (3 months) rats, and isometric force levels were lower in old rats. Both young and old rats displayed an increase in MyoD labeling in the exposed muscle, indicating that injury induced satellite-cell activation. However, there were more MyoD-labeled cells that coexpressed the proapoptotic factor, Bax, in old than in young rats, suggesting that decrements in muscle recovery may be associated with an increase in satellite-cell apoptosis. Based on these findings we conclude that reducing satellite-cell apoptosis in aged animals may improve muscle recovery after injury.

Transgenic Expression of Dominant Negative Tuberin through a Strong Constitutive Promoter Results in a Tissue-specific Tuberous Sclerosis Phenotype in the Skin and Brain

Tuberous sclerosis (TS) is a common autosomal dominant disorder caused by loss or malfunction of hamartin (tsc1) or tuberin (tsc2). Many lesions in TS do not demonstrate loss of heterozygosity for these genes, implying that dominant negative forms of these genes may account for some hamartomas and neoplasms in TS. To test this hypothesis, we expressed a dominant negative allele of tuberin (DeltaRG) behind the cytomegalovirus promoter in NIH3T3 cells and transgenic mice. This allele binds hamartin but has a deletion in the C terminus of tuberin, leading to constitutive activation of rap1 and rab5/rabaptin. Expression of DeltaRG in NIH3T3 cells led to a strong induction of reactive oxygen species, induction of vascular endothelial growth factor, and malignant transformation in vivo. Expression of DeltaRG driven by the constitutive cytomegalovirus promoter led to high level expression in all murine tissues examined, including skin, kidney, liver, and brain. Surprisingly, mice expressing the DeltaRG transgene developed a fibrovascular collagenoma in the dermis, which closely resembles the Shagreen patch observed in human patients with TS. In addition, numerous small subpial collections of external granule cells in the cerebellum were observed, which may be the murine equivalent of subependymal giant cell astrocytomas or tubers commonly seen in TS patients. Thus, expression of a dominant negative tuberin in multiple tissues can lead to a tissue-specific phenotype resembling some of the findings in human TS. Our data are the first to demonstrate that specific signaling abnormalities underlie specific hamartomas in a model of a human genetic disorder.

Analysis of Events Associated with Serum Deprivation-Induced Apoptosis in C3H/Sol8 Muscle Satellite Cells

Satellite cells are the source of new muscle fibers in postnatal skeletal muscle growth and regeneration. Regulation of satellite cell survival is of fundamental importance in maintaining normal muscle function. Here we describe and characterize a tissue culture model of satellite cell apoptosis. Kinetic studies indicate that serum deprivation triggers a set of sequential events: early cell death, transient cell cycle traverse, and delayed cell death. The satellite cell death occurs by apoptosis based on the internucleosomal DNA laddering,in situDNA end-labeling, and the requirements forde novoprotein synthesis and extracellular calcium influx. The transient period of cell cycle progression (7–11 h after serum withdrawal) is accompanied by temporal induction of members of the immediate early gene family, such as c-myc,c-fos,and SRF, and appears to precede the delayed phase of cell death. Satellite cell apoptosis can be suppressed by several growth factors and by blocking the activity of calpain, a calcium-regulated protease. The late phase of apoptosis is marked by selective activation of ubiquitin-mediated protein conjugation and degradation. This study defines several control points where satellite cell apoptosis may be genetically or pharmacologically intervened.